OBJECTIVE: We aimed to compare antiretroviral non-adherence in the behaviourally infected (BIAs) and congenitally infected adolescents (CIAs) and explore its associations with depression, cognitive impairment, and alcohol use disorder (AUD) in adolescents living with HIV(ALWHIV) in Botswana. METHODS: This study was a cross-sectional, comparative, multi-center research that involved collecting samples from different HIV clinics in Botswana. Of the 622 ALWHIV, 223 were identified as BIAs and 399 as CIAs. They were evaluated using various tools such as MINI-KID for psychiatric disorders, DSM-5 for AUD, CAT-rapid for cognitive assessment, and Visual Analogue Scale (VAS) for non-adherence (the outcome). The data were analysed using both bivariate and multivariate regression analyses. RESULTS: The participants' mean age (SD) was 17.7(1.60). The CIAs were more likely to have cognitive impairment (t -7.25; p < 0.01), while the BIAs had more depression (χ2 = 5.86; p = 0.016) and AUD (χ2 = 4.39; p = 0.036) and were more likely to be non-adherent (t = 3.14; p = 0.002). In the CIA group, cognitive impairment (AOR = 2.86; 95% CI:1.77-4.64) (AOR = 2.79; 95%CI:1.73-4.48) and depression (AOR = 2.69; 95%CI:1.48-4.90 were associated with ART non-adherence. In the BIA group, depression (AOR = 2.55; 95%CI:1.27-5.16), AUD (AOR = 2.58; 95%CI:1.21-5.49) and struggling to accept status (AOR = 2.54; 95%CI:1.41-4.56) predicted non-adherence to treatment. CONCLUSION: The two groups of adolescents differ regarding ART non-adherence and associated psychosocial issues, indicating the need for differentiated care to address non-adherence in the ALWHIV, especially in high-burden, resource-constrained settings, such as Botswana.