Previous research has shown that hyperactivation in ventral medial prefrontal cortex (VmPFC) and rostral anterior cingulate cortex (rACC) and high cortisol to corticotrophin ratio (cort:ACTH ratio) during neutral-relaxed states predict relapse in alcohol-dependent (AD) patients. Other studies have shown that VmPFC/rACC deactivation and blunted cortisol release to stress and alcohol cues are predictive of time to relapse and relapse severity. However, no previous study has assessed the relationship between these markers of central and peripheral nervous system dysfunction in AD participants and their potential joint effects on relapse risk. Forty early abstinent, treatment engaged AD patients underwent a laboratory experiment with exposure to neutral, alcohol and stress cues and a separate functional magnetic resonance imaging scan with similar cue exposure. Neutral-relaxed state cort:ACTH ratio was significantly associated with VmPFC hyperreactivity to neutral-relaxing cues, and also with hypoactivation in response to alcohol and stress cues in AD patients. Basal heart rate, neutral cort:ACTH ratio and neutral VmPFC hyperreactivty were each associated with risk of relapse. However, abnormal VmPFC activation and elevated cort:ACTH ratio overlap in predicting risk for relapse, and dysfunctional VmPFC response was the sole significant predictor of odds of relapse in a joint model of relapse risk. These findings suggest that the cort:ACTH ratio may serve as a peripheral marker of VmPFC brain dysfunction, while aberrant VmPFC responses need further evaluation as a potential biomarker of alcohol relapse risk in clinical outcome studies.