Evidence from physiological studies has been integral in many causal theories of behavioral and emotional problems. However, this evidence is hampered by the heterogeneity characterizing these problems. The current study adds to prior work by identifying neuro-physiological markers associated with heterogeneity in conduct problems (CP), callous-unemotional (CU) traits, and anxiety. Participants were classified into the following groups: (a) low risk, (b) anxious (predominately high anxiety), (c) primary (scored high on CP and CU traits but low on anxiety), and (d) secondary (high anxiety, CU traits, and CP). Developmental differences were also examined by including two different samples assessed during young adulthood (Study 1: n = 88; Mage = 19.92; 50% female) and childhood (Study 2: n = 72; Mage = 5.78, SD = 1.33; 39 males). Participants in both studies were recruited from community samples (Study 1: n = 2,306; Mage = 16, SD = .89; Study 2: n = 850; Mage = 5.01, SD = .95). Physiological responses (heart rate, skin conductance, startle modulation) were recorded while children and adults watched negative affective and neutral scenes. Medial prefrontal activation (oxygenated hemoglobin) was also measured in young adults. Findings suggested that individuals in the secondary and anxious psychopathy groups showed higher physiological arousal and startle reactivity to violent, fearful, and anger stimuli compared to individuals in the primary psychopathy group. In contrast, primary and secondary psychopathy groups showed similar physiological reactions to sad stimuli assessed during childhood. Also, young adults in the primary and secondary subtypes showed lower medial prefrontal cortex activation to violent stimuli compared to the anxious group. These findings provide evidence for the value of a multidomain approach for identifying neurophysiological mechanisms that can inform prevention and treatment efforts.