Background Most people who meet the diagnostic criteria for anti‐social personality disorder (ASPD) do not meet the criteria for psychopathy. A differentiating feature is affective‐interpersonal style. Eye blink startle reflex paradigms have been used to study affect. Aim The aim of this study is to explore an eye blink startle paradigm as a means of distinguishing between men with both ASPD and psychopathy, and men with ASPD alone. Methods One hundred and thirty‐six men were recruited as follows: 31 patients with ASPD and a Psychopathy Checklist‐Revised (PCL‐R) score of 26 or more, 22 patients with ASPD and a PCL‐R score of 25 or less, 50 forensic hospital employees and 33 general population men, none in the latter two groups having abnormal personality traits. Each was presented with 16 pleasant, 16 unpleasant and 16 neutral pictures. Acoustic probes were presented during each category at 300, 800, 1300 and 3800 milliseconds (ms) after picture onset. Eye blink response was measured by electromyography. Results Overall, both patient groups showed significantly smaller eye blink responses to the startle stimuli compared with the community controls. Both the latter and the ASPD group showed the expected increase in eye blink response at longer startle latencies to unpleasant pictures than pleasant pictures, but this was not present either in the group with psychopathy or in the forensic hospital employees. With increasing startle latency onset, eye blink amplitude increased significantly in both the healthy comparison groups and the ASPD group, but not in the group with psychopathy. Conclusions We replicated eye blink startle modulation deficiencies among men with psychopathy. We confirmed that the psychopathy and ASPD groups could be distinguished by startle stimulus onset asynchrony, but this pattern was also seen in one healthy group – the forensic hospital employees. This suggests a case for more research with more diverse comparison groups and more differentiation of personality traits before drawing definitive conclusions about distinctive startle response patterns among men with psychopathy. Copyright © 2014 John Wiley & Sons, Ltd.