ObjectivesIn 2017, Myanmar implemented routine viral load (VL) monitoring for assessing the response to antiretroviral therapy (ART) among people living with HIV (PLHIV). The performance of routine VL testing and implementation challenges has not yet assessed. We aimed to determine the uptake of VL testing and factors associated with it among PLHIV initiated on ART during 2017 in ART clinics of Yangon region and to explore the implementation challenges as perceived by the healthcare providers.DesignAn explanatory mixed-methods study was conducted. The quantitative component was a cohort study, and the qualitative part was a descriptive study with in-depth interviews.SettingSix ART clinics operated by AIDS/sexually transmitted infection teams under the National AIDS Programme.Primary outcome measures(1) The proportion who underwent VL testing by 30 March 2019 and the proportion with virological suppression (plasma VL <1000 copies/mL); (2) association between patient characteristics and ‘not tested’ was assessed using log binomial regression and (3) qualitative codes on implementation challenges.ResultsOf the 567 PLHIV started on ART, 498 (87.8%) retained in care for more than 6 months and were eligible for VL testing. 288 (57.8%, 95% CI: 53.3% to 62.2%) PLHIV underwent VL testing, of which 263 (91.3%, 95% CI: 87.1% to 94.4%) had virological suppression. PLHIV with WHO clinical stage 4 had significantly higher rates of ‘not being tested’ for VL. Collection of sample for VL testing only twice a month, difficulties in sample collection and transportation, limited trained workforce, wage loss and out-of-pocket expenditure for patients due to added visits were major implementation challenges.ConclusionsThe VL test uptake was low, with only six out of ten PLHIV tested. The VL testing uptake needs to be improved by strengthening sample collection and transportation, adopting point-of-care VL tests, increasing trained workforce, providing compensation to patients for wage loss and travel costs for additional visits.