Background: Previous research has suggested that adult psychopathic behavior and child callous-unemotional (CU) traits are uniquely related to low emotional reactivity. Salivary cortisol is a promising biological measure of emotional reactivity that has been relatively overlooked in research on CU traits and antisocial behavior. The current study examined for gender differences in the relation between resting salivary cortisol levels and CU traits in a non-referred adolescent sample. Salivary testosterone levels were assessed to provide discriminant validity for cortisol analyses and were not expected to bear a relation to CU traits. Method: An extreme groups strategy was used to recruit 108 adolescents (53 male, 55 female) from a larger screening sample who exhibited various combinations of low and high scores on parent-report measures of CU traits and conduct problems. Resting saliva samples were assayed for cortisol and testosterone levels using a radioimmunoassay procedure. Results: Consistent with prediction, male participants exhibiting elevated CU traits were uniquely characterized by low cortisol levels relative to male comparison groups (p less than 0.05). Testosterone levels did not differentiate groups and no hormone effects were found for female participants. Conclusions: The current findings build upon recent research in suggesting that low cortisol may be a biological marker for male CU traits.