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Little empirical attention has focused on the effects risk and protective factors during early adolescence on long-term trajectories of substance use. Moreover, research has yet to focus on gender differences in the effects of these early adolescence experiences, despite the fact that some risk factors have been found to have differing effects on boys and girls. Drawing on prospective longitudinal data from the Project on Human Development in Chicago Neighborhoods (PHDCN), this study 1) employs Group-Based Trajectory modeling to identify latent developmental trajectories of substance use for separate samples of males and females, and 2) uses multilevel multinomial logistic regression to assess the effects of risk and protective factors from individual, peer, family, and neighborhood domains on trajectory group membership for males and females. Results reveal distinct trajectories of substance use for adolescent boys and girls, and identify risk and protective factors for trajectory group membership. Findings have implications for the study of substance use through development, and underscore the importance of examining behavior separately by gender.