The objective of this study was to determine the rates of virological failure (VF) and HIV drug resistance (HIVDR) amongst adolescents on antiretroviral Therapy (ART). A retrospectively designed study was conducted in 10 healthcare centers for adolescents living with HIV (ALHIV) in the two main cities of Cameroon (Yaoundé and Douala), from November 2018 to May 2019. Sociodemographic, clinical, therapeutic and laboratory parameters were collected from medical records. All enrolled ALHIV had viral load (VL) measurements following the national guidelines. All patients with a VL ≥ 1000 copies/ml were called to perform genotyping tests. The chi-square test was used to determine the factors associated with VF. Out of the 1316 medical records of ALHIV, we included 1083 ALHIV having a VL result. Among them, 276 (25.5%) were experiencing VF, and VF was significantly higher in ALHIV with suboptimal adherence (p<0.001), older adolescents (p<0.05), those who lived outside the city where they were receiving ART (p<0.006), severely immunocompromised (p<0.01) and started ART at infancy (p<0.02). Among the 45/276 (16.3%) participants with an available genotyping resistance testing (GRT) result, the overall rate of HIVDR was 93.3% (42/45). The most common mutations were K103N (n = 21/42, 52.3%) resulting in high-level resistance to Efavirenz and Nevirapine, followed by M184V (n = 20/42, 47.6%) and thymidine analog mutations (n = 15/42, 35.7%) associated with high-level resistance to Lamivudine and Zidovudine respectively. The high rate of VF and HIVDR among ALHIV regularly followed in health facilities in Cameroon highlights the need to develop interventions adapted to an adolescent-centered approach to preserve future ART options.