Background Adolescents living with HIV on antiretroviral therapy (ART) have worse treatment adherence, viral suppression, and mortality rates compared to adults. This study investigated factors associated with viral non-suppression among adolescents living with HIV in Cambodia. Methods A cross-sectional study was conducted in August 2016 among 328 adolescents living with HIV aged 15–17 years who were randomly selected from 11 ART clinics in the capital city of Phnom Penh and 10 other provinces. Clinical and immunological data, including CD4 count and viral load, were obtained from medical records at ART clinics. Adolescents were categorized as having achieved viral suppression if their latest viral load count was < 1000 ribonucleic acid (RNA) copies/mL. Multivariate logistic regression analysis was performed to identify factors independently associated with viral non-suppression. Results The mean age of the participants was 15.9 years (SD = 0.8), and 48.5% were female. Median duration on ART was 8.6 (interquartile range = 6.0–10.6) years. Of total, 76.8% of the participants had achieved viral suppression. After adjustment for other covariates, the likelihood of having viral non-suppression remained significantly lower among adolescents who were: older/aged 17 (AOR = 0.46, 95% CI 0.21–0.98), had been on ART for more than 9 years (AOR = 0.35, 95% CI 0.19–0.64), had most recent CD4 count of > 672 (AOR = 0.47, 95% CI 0.26–0.86), had a relative as the main daily caregiver (AOR = 0.37, 95% CI 0.17–0.80), and did not believe that there is a cure for AIDS (AOR = 0.40, 95% CI 0.21–0.75) compared to their reference group. The likelihood of having viral non-suppression also remained significantly higher among adolescents who had first viral load > 628 RNA copies/mL (AOR = 1.81, 95% CI 1.05–4.08) and among those who were receiving HIV care and treatment from an adult clinic (AOR = 2.95, 95% CI 1.56–5.59). Conclusions The proportion of adolescents living with HIV with viral suppression in this study was relatively high at 76.8%, but falls short of the global target of 90%. Programs targeting younger adolescents and adolescents in transition from pediatric to adult care with a range of interventions including psychosocial support and treatment literacy could further improve viral suppression outcomes.