P3 amplitude reduction (P3AR) is associated with risk for adolescent-onset pathological substance use (PSU). In this longitudinal study, data from over 1,100 adolescent twins were used to examine P3AR in relation to early adolescent onset PSU (i.e., by age 14), late adolescent onset PSU (i.e., ages 14–18), misuse of different classes of substances (PSU-nicotine, PSU-alcohol, PSU-illicit), degree of PSU comorbidity, and gender differences. P3 amplitude was recorded at age 14 from two midline electrodes during a visual oddball paradigm. PSU was defined as meeting criteria for any symptom of a substance use disorder assessed using semistructured clinical interviews. P3AR was associated with degree of drug class comorbidity, early adolescent onset PSU for all three substance classes, and late adolescent onset PSU for alcohol and illicit PSU. Gender differences in P3AR were not statistically significant. These findings provide further evidence that P3AR indexes a nonspecific diathesis for adolescent-onset PSU.