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Abstract Same-day initiation (SDI) of antiretroviral therapy (ART) has been recommended to improve ART programme outcomes in South Africa since August 2017. This study assessed implementation of SDI over time in two South African districts, describing the characteristics of same-day initiators and evaluating the impact of SDI on retention in ART care. Routine data were analysed for HIV-infected adults who were newly initiating ART in Johannesburg or Mopani Districts between October 2017 and June 2018. Characteristics of same-day ART initiators were compared to later initiators, and losses to follow-up (LTFU) to six months were assessed using Kaplan Meier survival analysis and multivariate logistic regression. The dataset comprised 32 290 records (29 964 from Johannesburg and 2 326 from Mopani). The overall rate of SDI was 40.4% (n = 13 038), increasing from 30.3% in October 2017 to 54.2% in June 2018. Same-day ART initiators were younger, more likely to be female and presented with less advanced clinical disease than those initiating treatment at later times following diagnosis (p<0.001 for all). SDI was associated with disengagement from care: LTFU was 30.1% in the SDI group compared to 22.4%, 19.8% and 21.9% among clients initiating ART 1–7 days, 8–21 days and ≥22 days after HIV diagnosis, respectively (p<0.001). LTFU was significantly more likely among clients in Johannesburg versus Mopani (adjusted odds ratio (aOR) = 1.43, p<0.001) and among same-day versus later initiators (aOR = 1.45, p<0.001), while increasing age reduced LTFU (aOR = 0.97, p<0.001). In conclusion, SDI has increased over time as per national guidelines, but there is serious concern regarding the reduced rate of retention among same-day initiators. Nevertheless, SDI may result in a net programmatic benefit provided that interventions are implemented to support client readiness for treatment and ongoing engagement in ART care, particularly among younger adults in large ART programmes such as Johannesburg. Introduction South Africa’s antiretroviral therapy (ART) programme for the treatment of human immunodeficiency virus (HIV) infection was launched in 2004. At the time, 4.1 million adults over the age of 15 years were living with HIV and within a decade, an additional 2.2 million adults were infected with the virus [1]. National ART guidelines evolved over this time to treat increasing numbers of HIV-infected clients and to provide improved care and treatment services. South Africa’s ART programme has since grown to be the largest world-wide, with an estimated 4.2 million adults receiving ART in 2017 [2]. When South Africa’s ART programme first started, adults with CD4 counts <200 cells/mm3 were eligible to initiate treatment [3]. The CD4 cut-off was raised to 350 cells/mm3 in August 2011 [4] and to 500 cells/mm3 from January 2015 [5]. However, despite the expanding ART eligibility criteria, South Africa faced challenges in reaching the second 90-90-90 target, namely the provision of ART to 90% of HIV diagnosed individuals, with only 56.9% of HIV-diagnosed adults receiving ART nationally by the middle of 2015 [6]. In September 2016 the National Department of Health (NDoH) implemented universal test and treat (UTT) whereby all adults became eligible to initiate ART irrespective of CD4 count [7], but adult ART coverage increased only minimally to 61% in 2017 [2]. This is consistent with findings from a South African study regarding the significantly decreased likelihood of ART initiation with increasing baseline CD4 count [8], as clients feel “too healthy” to initiate treatment [9]. In addition, numerous other barriers to ART initiation may have impacted treatment coverage rates, including distance to testing centres, transport costs, over-busy clinics and the need for repeat facility visits at the time ART eligibility was being assessed [10, 11]. In order to address ongoing challenges with ART initiation, the NDoH implemented same-day initiation (SDI), namely ART initiation on the same day as HIV diagnosis [12]. The NDoH circular signed in August 2017 requested all public health facilities to scale up ART initiation for all HIV-infected individuals as per UTT guidelines, with an emphasis on providing SDI for individuals newly diagnosed with HIV who were clinically and psychologically ready for lifelong ART [12]. Clinical readiness encompasses screening for symptoms of tuberculosis (TB) and cryptococcal meningitis, as initiation of ART must be delayed in clients with these conditions in order to avoid complications such as immune reconstitution inflammatory syndrome [13]. When appropriately implemented, SDI has the potential to prevent the loss of ART-eligible clients from pre-ART care prior to treatment initiation, a challenge that has been described in multiple sub-Saharan African settings, including South Africa [14, 15]. A number of randomised trials of rapid ART initiation have indeed demonstrated multiple benefits of rapid initiation compared to later ART start, including improved ART uptake by 3 months, increased retention in care at 12 months, higher rates of 12-month viral suppression and reduced risk of mortality [16–19]. However, randomised trials precisely control the environment in which the intervention is being assessed, and it therefore cannot be automatically assumed that findings from these trials regarding the benefits of SDI would translate into routine settings [20]. Although a number of observational studies of SDI in sub-Saharan African settings have also been performed, these studies focussed on pregnant and breastfeeding women and the results may therefore not be generalizable to the wider adult population [21–24]. Other routine studies have been limited to hospitals [25] or have described SDI in the context of specialised interventions such as peer-delivered linkage case management or a revised ART initiation counselling model implemented at a single facility [26, 27]. There is thus a paucity of data regarding the implementation of a national policy for SDI across the whole adult population in routine sub-Saharan African settings. In order to fill this gap, this study aimed to analyse routine programme data to assess implementation of SDI over time and its impact on programme outcomes in an urban and rural district of South Africa. Specifically, this study aimed to compare characteristics of clients who initiate ART on the same day as HIV diagnosis to clients who initiate treatment at later time points after diagnosis, and to assess