Although chronic relapse is a characteristic of addiction to stimulants, conventional measures (eg, clinical, demographic, and self-report) do not robustly identify which individuals are most vulnerable to relapse. To test whether drug cues are associated with increased mesolimbic neural activity in patients undergoing treatment for stimulant use disorder and whether this activity is associated with risk for subsequent relapse. This prospective cohort study of 76 participants included a control group for baseline group comparisons. Veteran patients (n = 36) with stimulant use disorders were recruited from a 28-day residential treatment program at the Veterans Affairs Palo Alto Health Care System. Healthy controls (n = 40) were recruited from the surrounding community. Baseline data were collected between September 21, 2015, and January 26, 2018, from patients and healthy controls using functional magnetic resonance imaging during a performance of a reward cue task. Patients' stimulant use was subsequently assessed after treatment discharge (at approximately 1, 3, and 6 months) to assess relapse outcomes. Primary measures included neural responses to drug and food cues in estimated mesolimbic volumes of interest, including the medial prefrontal cortex, nucleus accumbens (NAcc), and ventral tegmental area. The primary outcome variable was relapse (defined as any stimulant use), assessed both dichotomously (3 months after discharge) and continuously (days to relapse). Brain activity measures were contrasted between groups to validate neural measures of drug cue reactivity, which were then used to estimate relapse outcomes of patients. Relative to controls (n = 40; 16 women and 24 men; mean SD age, 32.0 11.6 years), patients (n = 36; 2 women and 34 men; mean SD age, 43.4 13.3 years) showed increased mesolimbic activity in response to drug cues (medial prefrontal cortex, t74 = 2.90, P = .005, Cohen d = 0.66; NAcc, t74 = 2.39, P = .02, Cohen d = 0.54; and ventral tegmental area, t74 = 4.04, P < .001, Cohen d = 0.92). In patients, increased drug cue response in the NAcc (but not other volumes of interest) was associated with time to relapse months later (Cox proportional hazards regression hazard ratio, 2.30; 95% CI, 1.40-3.79). After controlling for age, NAcc response to drug cues classified relapsers (12 patients; 1 woman and 11 men; mean SD age, 49.3 14.1 years) and abstainers (21 patients; 1 woman and 20 men; mean SD age, 39.3 12.3 years) at 3 months with 75.8% classification accuracy. Model comparison further indicated that NAcc responses to drug cues were associated with relapse above and beyond estimations of relapse according to conventional measures. Responses in the NAcc to stimulant cues appear to be associated with relapse in humans. Identification of neural markers may eventually help target interventions to the most vulnerable individuals.