BACKGROUND: Despite the growing number of people on antiretroviral therapy (ART), there is limited information about virological non-suppression and its determinants among HIV-positive (HIV+) individuals enrolled in HIV care in many resource-limited settings. We estimated the proportion of virologically non-suppressed patients, and identified the factors associated with virological non-suppression. METHODS: We conducted a descriptive cross-sectional study using routinely collected program data from viral load (VL) samples collected across the country for testing at the Central Public Health Laboratories (CPHL) in Uganda. Data were generated between August 2014 and July 2015. We extracted data on socio-demographic, clinical and VL testing results. We defined virological non-suppression as having ≥1000 copies of viral RNA/ml of blood for plasma or ≥5000 copies of viral RNA/ml of blood for dry blood spots. We used logistic regression to identify factors associated with virological non-suppression. RESULTS: The study was composed of 100,678 patients; of these, 94,766(94%) were for routine monitoring, 3492(4%) were suspected treatment failures while 1436(1%) were repeat testers after suspected failure. The overall proportion of non-suppression was 11%. Patients on routine monitoring registered the lowest (10%) proportion of non-suppressed patients. Virological non-suppression was higher among suspected treatment failures (29%) and repeat testers after suspected failure (50%). Repeat testers after suspected failure were six times more likely to have virological non-suppression (OR = 6.3, 95%CI = 5.5-7.2) when compared with suspected treatment failures (OR = 3.3, 95%CI = 3.0-3.6). The odds of virological non-suppression decreased with increasing age, with children aged 0-4 years (OR = 5.3, 95%CI = 4.6-6.1) and young adolescents (OR = 4.1, 95%CI = 3.7-4.6) registering the highest odds. Poor adherence (OR = 3.4, 95%CI = 2.9-3.9) and having active TB (OR = 1.9, 95%CI = 1.6-2.4) increased the odds of virological non-suppression. However, being on second/third line regimens (OR = 0.86, 95%CI = 0.78-0.95) protected patients against virological non-suppression. CONCLUSION: Young age, poor adherence and having active TB increased the odds of virological non-suppression while second/third line ART regimens were protective against non-suppression. We recommend close follow up and intensified targeted adherence support for repeat testers after suspected failure, children and adolescents.