A comprehensive genetic diversity analysis of 87 Passiflora germplasm accessions domesticated and cultivated for several years in the karst region of Guizhou, China, was conducted utilizing simple sequence repeat (SSR) fluorescent markers. These Passiflora species, renowned for their culinary and medicinal value, could bring significant economic and ecological benefits to the region. This study aimed to assess the genetic resources of these species and facilitate the selection of superior cultivars adapted to the karst environment. Our analysis revealed an abundance of SSR loci within the Passiflora transcriptome, with single-base repeats being the most prevalent type. Through rigorous primer screening and amplification, we successfully identified 27 SSR primer pairs exhibiting robust polymorphisms. Further interrogation at eight microsatellite loci revealed 68 alleles, underscoring the high level of genetic diversity present in the cultivated accessions. The average expected heterozygosity was 0.202, with the ssr18 locus exhibiting the highest value of 0.768, indicating significant genetic variation. The mean polymorphic information content (PIC) of 0.657 indicates the informativeness of these SSR markers. Comparative analyses of the cultivated and potential wild progenitors revealed distinct genetic variations among the different Passiflora types. Genetic structure and clustering analyses of the 87 accessions revealed seven distinct groups, suggesting gene flow and similarities among the resources. Notably, a DNA fingerprinting system was established using eight SSR primer pairs, effectively distinguishing the selected cultivars that had adapted to the karst mountainous region. This study not only deepens our understanding of Passiflora genetic resources in the karst environment but also provides a valuable reference for conservation, genetic improvement, and cultivar selection. The rich genetic diversity of the Passiflora germplasm underscores their potential for sustainable utilization in breeding programs aimed at enhancing the economic and ecological viability of these valuable plant species.