Although roughly 70% of the American Indian and Alaska Native (AI) population live in urban areas, research is scarce regarding this population. As a consequence, there is limited understanding about the salient socioenvironmental factors that aid in preventing substance use among urban AI communities. This study utilized a statewide, cross-sectional, school-based survey of urban AI adolescents (N = 2,375) to (a) examine the associations between substance use and risk and promotive factors within the family and peer group, and (b) explore how these associations vary by subgroups (gender, racial/ethnic background, and grade level). Results suggest that risk factors-familial substance use and antisocial peer affiliation-were associated with higher alcohol, cigarette, and marijuana use. However, these findings varied by subgroup. For males, involvement with antisocial peers was associated with greater marijuana use. Involvement with antisocial peers was also positively associated with alcohol and marijuana use for multiracial/multiethnic AI adolescents and those adolescents in 10th and 12th grades. The promotive factors-supportive family environment and prosocial peer affiliation-were not universally associated with lowered substance use by subgroup. This study advances understandings of the risk and promotive factors important in reducing and preventing substance use among urban AI adolescents. Experiencing familial substance use and affiliating with antisocial peers were the salient factors associated with increased substance use, particularly for urban AI adolescents who are older, male, and with multiracial/multiethnic AI backgrounds.