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Background: In Africa, data on viral suppression among human immunodeficiency virus (HIV)-positive children and adolescents on antiretroviral (ART) are scarce. This study aimed to determine determinants of HIV viral load (VL) non-suppression among HIV-positive children and adolescents (< 20 years old) who attend care and treatment clinics (CTCs) in Tabora region from January 2018 to April 2022. Data were abstracted from CTCs' database and patient records including socio-demographic, ART drug regimen, clinical, virological and immunological features, for a maximum of 52 months. Viral non-suppression was defined as plasma VL ≥ 1000 copies/mil after 6 months on ART, during the follow-up period. Cox proportional hazards regression model and hazard ratios (HRs) were used in estimating the determinants of viral non-suppression. Results: A total of 378 HIV-positive children (0–9 years) and adolescents (10–19 years) were enrolled, of whom 124 (32.8%) had virological non-suppression. The overall rate of VL non-suppression was 1.38 (95% CI 1.15, 1.64) per 100 person-months of observation. After adjusting for other factors, determinants of viral non-suppression were poor ART adherence level at initiation of ART (HR = 3.3; 95% CI 2.16, 4.91), low CD4 count at ART initiation (HR = 1.66; 95% CI 1.20, 2.30), nevirapine (NVP)-based regimen (HR = 2.64; 95% CI 1.32, 5.26), efavirenz (EFV)-based regime (HR = 2.08; 95% CI 1.03, 4.18), lopinavir/ritonavir (LPV/r)-based regimen (HR = 2.21; 95% CI 1.13, 4.32) and being on second-line regimen (HR = 6.11; 95% CI 2.50, 14.96). Conclusions: HIV viral non-suppression among children and adolescents on ART in central Tanzania in the Tabora region is high (32.8%) and is associated with poor ART adherence level, low CD4 count, NVP-, EFV-, and LPV/r-based regimen. Early initiation of ART and intensified monitoring are required to improve viral suppression rates of HIV-infected children to attain the third goal of the UNAIDS 95-95-95. [ABSTRACT FROM AUTHOR]