Background Substance use among people living with HIV is associated with poor health, social, and psychological outcomes. This study assessed the prevalence of substance use and associated factors among youth attending HIV care and treatment centers (CTCs) in Dodoma, Tanzania. Methods This cross-sectional study was carried out in Dodoma, Tanzania, from February to April 2020 among youth aged 15–24 attending HIV CTCs. Data was collected using sociodemographic, WHO ASSIST Version 3.0, BDI II, and SERAD questionnaires. Data analysis was done using Stata 17. Descriptive statistics were used to summarize continuous and categorical variables. Univariable and multivariable logistic regression analyses were conducted to determine factors independently associated with substance use. Results The prevalence of substance use was relatively low (6.6%). Older youth (20 to 24 years) were 2% less likely to use substances compared to the younger ones (15 to 19 years) (AOR: 0.07; 95% CI 0.01, 0.83). There were statistically significant decreasing odds of substance use with every year increase in age at HIV diagnosis (OR: 1.66; 95% CI 1.14, 2.41). Being unemployed was statistically significantly associated with decreased odds of substance use among this population (OR: 0.03; 95% CI 0.02, 0.33). Youth who had detectable viral loads were significantly more likely to use substances compared to those with undetectable viral loads (AOR: 12.9; 95% CI 1.07, 156.05). Conclusions Despite the low prevalence of substance use found in this study, it is important to note that late age of HIV diagnosis, employment, and detectable viral load negatively impacted HIV positive youth with regards to substance use. It is recommended that CTCs emphasize routine screening for substance use among youth who have detectable viral loads.