Background: A robust and bi-directional comorbidity between attention-deficit/hyperactivity disorder (ADHD) and psychoactive substance use disorder (PSUD, alcohol or drug abuse, or dependence) has been consistently reported in the literature. However, this literature has been based almost exclusively on male only samples and, therefore, the findings may not generalize to females. Methods: First-degree relatives from a large sample of pediatrically and psychiatrically referred girls with (123 probands, 403 relatives) and without ADHD (112 probands, 359 relatives) were comprehensively assessed by blind raters with structured diagnostic interviews. Familial risk analysis examined the risks in first-degree relatives for ADHD and PSUD (alcohol or drug abuse or dependence) after stratifying probands by the presence and absence of these disorders. Results: ADHD in the proband significantly increased the risk for ADHD in relatives independently of the comorbidity with PSUD. PSUD in the proband was associated with a significantly increased risk for PSUD in relatives regardless of ADHD status. There was no evidence of co-segregation or non-random mating in the families of probands with ADHD and PSUD. Conclusions: Patterns of familial risk analysis suggest that the association between ADHD and PSUD in adolescent females is most consistent with the hypothesis that these disorders are independently transmitted, although the hypothesis of variable expressivity could not be ruled out. These findings are consistent with previously reported patterns of familial associations between ADHD and PSUD found in adolescent males. Longer follow-up periods are needed to more fully clarify the relationship between ADHD and PSUD, as well as provide adequate power for separate analyses of alcohol and drug use.