This study evaluated effects of the Iowa Strengthening Families Program, a family-focused universal preventive intervention, on growth patterns of adolescent internalizing (anxiety and depressive symptoms) and monthly polysubstance use (alcohol, tobacco, marijuana, inhalants, and other illicit drugs), as well as the association between internalizing and polysubstance growth factors. The sample consisted of rural Midwestern adolescents (N = 383), followed from sixth through twelfth grade. Compared to the control group, the intervention group adolescents showed a slower rate of increase in internalizing symptoms and polysubstance use. Intervention effects on internalizing symptoms were similar for boys and girls; however, girls demonstrated a higher overall level and a greater rate of increase across time. The intervention slowed the rate of increase in polysubstance use significantly more for girls than for boys, although overall levels of use were lower in the intervention group for both genders. Associations between internalizing and polysubstance use growth factors were found for girls, but not for boys, suggesting gender differences in psychosocial development.