Sexual and gender minority individuals assigned female at birth (SGM-AFAB) are at increased risk for anxiety, depression, and substance use and problems compared with heterosexual cisgender women. Cross-sectional research has demonstrated that minority stressors are associated with anxiety, depression, and substance use. However, longitudinal research is limited and the examination of prospective associations between minority stressors, mental health, and substance use is even more sparse. We utilized 4 waves of data (6 months between waves) from a diverse (26.0% non-Latinx White; 26.2% gender minorities) longitudinal cohort of 488 SGM-AFAB (16- to 32-years-old at Wave 1) to examine concurrent and prospective associations between 3 minority stressors (internalized stigma, microaggressions, victimization) and anxiety, depression, and alcohol and cannabis use and problems. At the within-person level, results indicated that when SGM-AFAB experienced more minority stressors than usual, they reported more concurrent and prospective anxiety and depression. Additionally, when SGM-AFAB experienced more microaggressions than usual, they were more likely to use alcohol and cannabis, and when they experienced more victimization than usual, they reported more concurrent alcohol and cannabis use problems. No prospective associations between minority stressors and substance use were significant. Findings indicate that minority stressors were consistently associated with internalizing symptoms, both concurrently and prospectively, while evidence for associations between minority stressors and substance use/problems was less consistent. These findings highlight the need for interventions that that teach SGM-AFAB skills for effectively coping with minority stress. (PsycInfo Database Record (c) 2020 APA, all rights reserved).