We report here the genetic characterization of bitter gourd (Momordica charantia) based on polymorphisms of 50 simple sequence repeat (SSR) loci in 114 accessions that included landraces, breeding lines, and commercial open-pollinated and F1 hybrid cultivars widely grown in Asia. Neighbor-joining tree analysis revealed a high level of genetic variability in the collection. The 114 accessions formed three subpopulations represented by five clusters. Distribution of accessions across the five clusters reflected their geographic origin to a large extent. South Asian accessions originating from India, Bangladesh, and Pakistan were more closely related to each other than to any other geographical group. Likewise, southeast Asian accessions that originated from Cambodia, Vietnam, Indonesia, and Philippines were grouped together. Accessions that originated from Taiwan were genetically distinct and grouped separately. A landrace from Laos was genetically close to the accessions from Thailand and genetically distinct from the rest of the accessions. White-fruited genotypes were genetically distinct from green- and dark green–fruited genotypes. Low- and medium-bitter accessions were more similar to each other than to the high-bitter genotypes. Accessions with cylindrical fruit were genetically distinct from those with spindle or elongated fruit. Commercial cultivars in each cluster were closely related, which indicated a narrowing of the bitter gourd genetic base in Asia in response to market demands for uniformity and yield. Use of diverse germplasm resources in bitter gourd breeding will help in sustainable breeding and production.