Background: Risks associated with parental separation have received limited attention in research on children of parents with substance use disorders. We examined early substance involvement as a function of parental separation during childhood and parental alcohol and cannabis dependence. Method: Data were drawn from 1,318 adolescent offspring of monozygotic (MZ) or dizygotic (DZ) Australian twin parents. Cox proportional hazards regression analyses were conducted predicting age at first use of alcohol, first alcohol intoxication, first use and first regular use of cigarettes, and first use of cannabis, from parental separation and both parent and cotwin substance dependence. Parent and cotwin alcohol and cannabis dependence were initially modeled separately, with post-hoc tests for equality of effects. Results: With few exceptions, risks associated with parental alcohol versus cannabis dependence could be equated, with results largely suggestive of genetic transmission of risk from parental substance (alcohol or cannabis) dependence broadly defined. Controlling for parental substance dependence, parental separation was a strong predictor for all substance use variables, especially through age 13. Conclusion: Together, findings underscore the importance of parental separation as a risk-factor for early substance involvement over and above both genetic and environmental influences specific to parental alcohol and cannabis dependence.