The current study investigated genetic and environmental influences on salivary testosterone during adolescence, using data from 49 pairs of monozygotic twins and 68 pairs of dizygotic twins, ages 14–19 years (M = 16.0 years). Analyses tested for sex differences in genetic and environmental influences on testosterone and its relation to pubertal development. Among adolescent males, individual differences in testosterone were heritable (55%) and significantly associated with self-reported pubertal status (controlling for age) via common genetic influences. In contrast, there was minimal heritable variation in testosterone for females, and testosterone in females was not significantly associated with pubertal status after controlling for age. Rather, environmental influences shared by twins raised together accounted for nearly all of the familial similarity in female testosterone. This study adds to a small but growing body of research that investigates genetic influences on individual differences in behaviorally relevant hormones. © 2014 Wiley Periodicals, Inc. Dev Psychobiol 56: 1278–1289, 2014.