OBJECTIVES: This study sought to examine the prevalence of STIs and whether substance use frequency and/or problematic use-specifically alcohol, marijuana and other drugs-was associated with having an STI diagnosis among youth living with HIV (YLWH) METHODS: A sample of 823 YLWH were recruited at 14 adolescent HIV clinics through the Adolescent Medicine Trials Network for HIV Interventions. Study staff abstracted STI data from medical records for up to 26 weeks prior to participants' completing a cross-sectional survey including the ASSIST (Alcohol, Smoking and Substance Involvement Screening Test), which measures substance use frequency and consequences. RESULTS: Almost one-third of youth had been diagnosed with an STI (30.5%) at the time of their baseline assessment. In multivariable analyses, those who engaged in weekly or greater marijuana use (adjusted OR (AOR)=10.66, 95% CI: 4.39 to 25.87, P<0.001) had an increased odds of being diagnosed with an STI. Additionally, youth who met alcohol use criteria for moderate (AOR=5.23, 95% CI: 2.50 to 10.93, P<0.001) and high risk (AOR=6.53, 95% CI: 1.20 to 35.68, P<0.05) alcohol use had an increased odds of being diagnosed with an STI compared with low-risk alcohol users. CONCLUSIONS: Study findings underscore the need to investigate the role of greater frequency of marijuana use and problematic alcohol use in STI incidence among YLWH. Given the associations between both substance use frequency and problematic use in STI diagnoses among YLWH seen in HIV care settings, clinicians should use validated substance use screening tools which capture both frequencies and consequences in order to identify YLWH who may need further evaluation and treatment.