Most individuals will gamble during their lifetime, yet only a select few will develop gambling disorder. Gray’s Reinforcement Sensitivity Theory holds promise for providing insight into gambling disorder etiology and symptomatology as it ascertains that neurobiological differences in reward and punishment sensitivity play a crucial role in determining an individual’s affect and motives. The aim of the study was to assess a mediational pathway, which included patients’ sex, personality traits, reward and punishment sensitivity, and gambling-severity variables. The Sensitivity to Punishment and Sensitivity to Reward Questionnaire, the South Oaks Gambling Screen, the Symptom Checklist-Revised, and the Temperament and Character Inventory-Revised were administered to a sample of gambling disorder outpatients (N = 831), diagnosed according to DSM-5 criteria, attending a specialized outpatient unit. Sociodemographic variables were also recorded. A structural equation model found that both reward and punishment sensitivity were positively and directly associated with increased gambling severity, sociodemographic variables, and certain personality traits while also revealing a complex mediational role for these dimensions. To this end, our findings suggest that the Sensitivity to Punishment and Sensitivity to Reward Questionnaire could be a useful tool for gaining a better understanding of different gambling disorder phenotypes and developing tailored interventions.