Background Altered levels of cortisol and testosterone have previously been associated with anti‐social personality disorder (ASPD) and psychopathy, but there is some conflicting evidence as to how characteristic these findings are. Aim To test the hypothesis that diurnal fluctuations in cortisol and/or testosterone will differentiate ASPD and psychopathy among male forensic psychiatric inpatients and distinguish both groups from healthy men not in treatment. Methods One hundred and sixty‐six men participated: 81 patients with ASPD, 42 of whom had a Psychopathy Checklist‐Revised (PCL‐R) score of 26 or more and 39 with a score of 25 or less, 51 forensic hospital employees and 34 general population men. None in the latter two groups had abnormal personality traits. For each person, diurnal cortisol and testosterone saliva samples were collected. Results Both patient groups and the forensic hospital employees showed significantly higher diurnal testosterone levels than the general population, community‐based men. The community men showed significantly lower values in their diurnal cortisol variation than the ASPD and psychopathy groups but, in this, were similar to the forensic employee group. Neither cortisol nor testosterone levels differentiated the higher from lower Psychopathy Checklist‐Revised scorers. Conclusions We replicated findings of diurnal testosterone deficiencies among men with psychopathy and ASPD, but we were unable to differentiate patients groups from each other or from the hospital employees on cortisol measures. This suggests a case for more research with more diverse comparison groups and more differentiation of personality traits before drawing definitive conclusions about distinctive hormonal patterns among men with psychopathy, as external environmental variables may prove more influential than previously suspected. Copyright © 2015 John Wiley & Sons, Ltd.