Lagenaria siceraria (Molina) Standley is an important cultivated crop with its immense importance in pharmaceutical industry and as vegetable. Its seed, root, stem, leaves, flower, and fruit are used as an ointment for ailment of various diseases throughout Asia. Despite its worldwide importance, informative co-dominant microsatellite markers in the bottle gourd crop are very restricted, impeding genetic improvement, cultivar identification, and phylogenetic studies. Next-generation sequencing has revolutionized the approaches for discovery, assessment, and validation of molecular markers. We conducted a genome-wide analysis, for developing SSR markers by utilizing restriction site-associated DNA sequencing (RAD-Seq) data obtained from NCBI. By performing in silico mining of microsatellite repeat motifs, we developed 45,066 perfect SSR markers. Of which 207 markers were successfully validated and 120 (57.97%) polymorphic primer pairs were utilized for an in-depth genetic diversity and population structure analysis of 96 accessions from the National Genebank of India. Tetranucleotide repeats (∼34.3%) were the most prevalent followed by trinucleotide repeats (∼30.73%), further 21.03%, 9.6%, and 4.3% of di-, penta-, and hexa-nucleotide repeats in the bottle gourd genome, respectively. Synteny of SSR markers on 11 bottle gourd linkage groups was correlated with the 7 chromosomes of cucumber (93.2%), 12 chromosomes of melon (87.4%), and 11 of watermelon (90.8%). The generated SSR markers provide a valuable tool for germplasm characterization, genetic linkage map construction, studying synteny, gene discovery, and for breeding in bottle gourd and other cucurbits species. KEY MESSAGE: Development of 45,066 perfect microsatellite markers as a valuable tool for marker assisted selection (MAS) in plant breeding.