The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) replaced the fourth edition's (DSM-IV) diagnosis of Pathological Gambling (PG) with Gambling Disorder (GD). GD differs from PG in that it requires 4 rather than 5 criteria for diagnosis and excludes the "Illegal Acts" criterion. We examined the prevalence of GD and its characteristics and validity in a substance-use disorder (SUD) sample. Participants (N = 6,613) in genetic studies of substance dependence underwent a semistructured psychiatric interview. Individuals who reported ever having gambled $10 at least monthly (n = 1,507) were the focus of the analyses. Approximately one third of acknowledged gamblers (n = 563; 8.5% of the total sample) received PG (DSM-IV) and GD (DSM-5) diagnoses and 678 (10.3% of the total) received only a DSM-5 diagnosis, representing an increase of 20.4% relative to DSM-IV. Although the 3 groups were comparable demographically, the DSM-5-Only group was intermediate between the other 2 groups on the prevalence of comorbid SUDs, the distribution of DSM-IV PG criteria endorsed, and the types of gambling reported. Multinomial logistic regression analysis showed that the DSM-5-Only group was more likely than the No-Diagnosis group and less likely than the Both-Diagnoses group to acknowledge a gambling problem. In conclusion, there was a high prevalence of PG in this SUD sample. Analysis of non-DSM variables suggested that the increased sensitivity of the DSM-5 GD diagnosis successfully identifies a broader set of individuals with clinically significant gambling-related problems. Prospective studies of individuals with GD are needed to validate this finding.