<p>Kitti’s hog-nosed bat or <i>Craseonycteris thonglongyai</i> is the only species in the family Craseonycteridae genus Craseonycteris and is considered as Endangered on the IUCN Red List of near-threatened vulnerable animals. This study aimed primarily to develop and characterize novel SSR markers specific to <i>C. thonglongyai</i> and secondarily to apply these markers for a preliminary assessment of genetic diversity within the species to develop a strategic conservation management plan. A total of 86 simple sequence repeats (SSR) primer pairs were developed for <i>C. thonglongyai</i> using an enriched-genomic DNA library with (GT)₁₂/(CTG)₉ and (AG)₁₂/(CAG)₉ probes. Of these, six loci showed polymorphisms within the tested <i>C. thonglongyai</i> samples. Genetic diversity estimates of 57 <i>C. thonglongyai</i> samples were based on 10 polymorphic SSR loci, including 6 newly developed loci from this study and 4 loci previously reported. In total, 48 alleles with an average of 4.8 alleles per locus were identified. The mean observed heterozygosity, expected heterozygosity, and polymorphic information content were 0.506, 0.486, and 0.494, respectively. Although no linkage disequilibrium was observed, three loci showed significant deviations from the Hardy–Weinberg equilibrium. Based on the analysis of 10 polymorphic SSR loci across 57 <i>C. thonglongyai</i> samples, the population in Thailand exhibited a moderate level of genetic diversity. The clustering observed in the phylogenetic tree and principal coordinate analysis did not correspond to the geographic locations of the caves. Moreover, cross-transferability of the polymorphic SSR loci from <i>C. thonglongyai</i> was successful in other bat species. Among the 10 polymorphic SSR loci tested across four bat families, 6 loci successfully amplified in at least one species of the Hipposideridae, Vespertilionidae, and Rhinolophidae, with polymorphic patterns observed in these amplified loci.</p>