Improved breeding for developing fruit quality in bottle gourd (Lagenaria siceraria (Mol.) Standl.) necessitates knowledge regarding its genetic diversity. To achieve this, a set of 108 locus-specific SSR markers has been developed in bottle gourd by cross-species transferability from 995 mapped Cucumis sativus SSR markers. During screening, 280 primer pairs amplified in the bottle gourd germplasm, which were further evaluated in a diverse set of 42 lines, resulting in 19 polymorphic, 89 monomorphic, 15 with multiple bands, and the rest 157 showed no or very non-specific amplification. The 19 polymorphic primer pairs produced a total of 54 alleles. Gene diversity, Shannon's information index, and Nei's coefficient of differentiation were calculated suggesting a moderate genetic variation at the species level. A model-based population structure analysis divided these germplasm into two subpopulations. This marker set will be applicable for evaluating the genetic structure for association mapping, DNA fingerprinting, and mounting linkage maps and will be a practical tool set for further genetics. This study provides one of the first quantitative views of population genetic variation in bottle gourd.