BACKGROUND: Adolescents are lagging behind in the "third 95" objective of the Joint United Nations Program on HIV/AIDS requiring 95% of individuals on antiretroviral therapy (ART) to have viral load (VL) suppression. This study aimed to describe factors associated with viral non-suppression among adolescents in Mbale district, Uganda. METHODS: We conducted a retrospective review of routinely collected HIV programme records. Data such as age, education, ART Regimen, ART duration, WHO Clinical stage, comorbidities, etc., were extracted from medical records for the period January 2018 to December 2018. Descriptive analysis was done for continuous variables using means and frequencies to describe study sample characteristics, and to determine the prevalence of outcome variables. We used logistic regression to assess factors associated with VL non-suppression among adolescents. RESULTS: The analysis included 567 HIV-infected adolescents, with 300 (52.9%) aged between 13 to 15 years, 335 (59.1%) female, and mean age of 15.6 years (interquartile range [IQR] 13.5-17.8. VL non-suppression was 31.4% (178/567). Male sex (AOR = 1.78, 95% CI 1.06, 2.99; p < 0.01), age 16-19 years (AOR = 1.78, 95% CI 1.06, 2.99; p < 0.05), No formal education (AOR = 3.67, 95% CI 1.48-9.09; p < 0.01), primary education (AOR = 2.23, 95% CI 1.05-2.32; p < 0.01), ART duration of > 12 months to 5 years (AOR = 3.20, 95% CI 1.31-7.82; p < 0.05), ART duration > 5 years (AOR = 3.47, 95% CI 1.39- 8.66; p < 0.01), WHO Clinical Stage II (AOR = 0.48, 95% CI: 0.28, 0.82; p < 0.01), second-line ART regimen (AOR = 2.38, 95% CI 1.53-3.72; p < 0.001) and comorbidities (AOR = 3.28, 95% CI 1.20-9.00; p < 0.05) were significantly associated with viral non-suppression. CONCLUSIONS: VL non-suppression among adolescents was almost comparable to the national average. VL non-suppression was associated with being male, age 16-19 years, education level, duration on ART therapy, WHO Clinical Staging II, second-line ART regimen, and presence of comorbidities. Adolescent-friendly strategies to improve VL suppression e.g. peer involvement, VL focal persons to identify and actively follow-up non-suppressed adolescents, patient education on VL suppression and demand creation for ART are needed, especially for newly-initiated adolescents and adolescents on ART for protracted periods, to foster attainment of the UNAIDS 95-95-95 targets.