ABSTRACT. Objective: This research examines gender and racial/ ethnic differences in substance use trajectories during early adolescence among American Indian and non-Native adolescents. Method: Substance use trajectories were evaluated among 684 adolescents (50% female, 51% American Indian) across five assessments over 9th and 10th grades. Youth were drawn from six rural towns within the Cherokee Nation, a nonreservation tribal jurisdiction that includes a high proportion of American Indians embedded within a predominantly White population. Past-month substance use was based on self-report and was dichotomized into "used" versus "did not use," with the exception of alcohol, which was trichotomized into "none," "1 or 2 days," or "3-30 days." Results: Using growth mixture modeling with full-information maximum-likelihood estimation, we determined that between two and three different trajectory classes best described the data for each substance. Males had a higher probability compared with females of following a trajectory of chewing tobacco use (20% vs. 6%, respectively) and using multiple substances (24% vs. 19%, respectively). Females had a higher probability compared with males of following a trajectory of prescription drug misuse (11% vs. 6%, respectively). Individuals who followed trajectories of alcohol use or heavy drinking were also more likely to follow trajectories of other substance use. Conclusions: Identifying gender and racial/ethnic differences in patterns of substance use at this stage of development will inform gender-sensitive and ethnically sensitive prevention programs targeting specific substance use. These results will be particularly informative given the lack of evidence regarding trajectories of substance initiation within largely American Indian populations. (J. Stud. Alcohol Drugs, 77, 238-248, 2016)