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Introduction South Africa has been reported to have a high burden of HIV and is currently running the largest anti-retroviral treatment (ART) programme globally which incorporates the test and treat policy. However, adherence to ART is particularly crucial for the optimal health outcomes of HIV patients. Evidence revealed that there are barriers to medication adherence in many parts of the country. Factors such as sociodemographic and patient’s characteristics have been found to be associated with ART adherence. The aim of this study was to measure the extent to which patients adhere to ART medication refill pick-up and to identify factors associated with adherence to ART medication refill pick-up among patients attending the regional hospital HIV clinics in Sekhukhune district, Limpopo province.Methods A retrospective cohort study of patients who initiated ART between August 2020 and August 2021 was conducted. TIER.net electronic data were reviewed and extracted into Microsoft Excel 2010. The extracted data were analysed using EPI-Info V.7.2.5.0 software. Factors such as patients’ age, gender, pregnancy, co-morbidity, baseline CD4 count, ART regimen, tuberculosis (TB), WHO clinical HIV stage, treatment duration and number of missed pharmacy refill pick-up appointments were collected.Results Among a total of 731 participants, only 30.5% of participants were found to be adherent to medication refill pick-up. Adherence to ART medication refill pick-up was the highest (56.0%) in facilities at Ephraim Mogale sub-district as compared with facilities in Elias Motsoaledi sub-district, with the OR of 0.359 and 95% CI of (0.243 to 0.532) with a significant p value (p=0.001). However, this study did not reveal a significant association between adherence to medication refill pick-up and other sociodemographic characteristics such as age, gender, pregnancy and TB.Conclusions Patients collecting medication from sub-districts with increased HIV patient influxes are less likely to be adherent to ART medication refill pick-up as compared with participants attending care from sub-districts with reduced HIV patient volume.