PROJECT SUMMARY/ABSTRACT Sexual minority (SM, i.e., people who are not heterosexual) and gender minority (GM, i.e., people who have a gender that is discordant from the sex they were assigned at birth, as opposed to cisgender people who have a gender that is concordant with the sex they were assigned at birth) people (collectively abbreviated as SGM) are at greater risk for health disparities including very high rates of substance use. Within studies on substance use, GM people are largely unrepresented as GM status is rarely measured or reported. The primary explanation for the higher rates of substance use and health problems among SGM people is minority stress, which confers an additional stress burden on SGM people including experiences of discrimination, expectations of discrimination, concealment of one’s identity, and internalization of social stigma. Epigenetic modifications (e.g., DNA methylation) are one way to track molecular modifications in response to substance use and minority stress. Understanding the epigenetics of substance use and minority stress may help us to develop better ways to identify and treat substance use disorders and to understand the downstream health outcomes of SGM people. This study will leverage The PRIDE Study, a national longitudinal cohort study of SGM people to identify trajectories in substance use among SGM people over 3 years of annual data collection (N > 3937 aged 18+), examine differences in these trajectories for GM versus cisgender SM people, and examine relationships between minority stress and substance use trajectories (Aim 1). This study will then identify the relationships between substance use trajectories and DNA methylation (N=600, half of whom are GM), identify differences in the relationships between substance use and DNA methylation for GM versus cisgender SM people, and identify which of these relationships between DNA methylation and substance use persist even after accounting for minority stress (Aim 2). This study will help us to understand longitudinal trajectories of substance use among SM and GM people to inform targeted prevention and intervention development. This study will also inform the development of biomarkers for substance use to improve substance use treatment and prevention.