Pitaya (Hylocereus spp.) is a tropical fruit grown in Okinawa prefecture, Japan. In this study, we developed simple sequence repeat (SSR) markers to assess the genetic diversity of pitaya accessions collected in Okinawa prefecture. SSR markers were developed with next-generation sequencing, using the Roche 454 GS FLX+ platform. Seventy primer sets were tested for the genetic identification of pitaya, including 50 AG motif and 20 AC motif SSRs. In total, 16 SSR markers successfully amplified fragments and exhibited polymorphism among the accessions. The SSR markers revealed the genetic diversity parameters including number of alleles per locus (ranging from 6 to 12), expected heterozygosity (ranging from 0.5 to 0.85), observed heterozygosity (ranging from 0.23 to 0.93), polymorphic information content (ranging from 0.47 to 0.83), fixation index value (0.013), and out-crossing rate (0.973). Using these SSR markers, genetic identification was performed for 32 pitaya accessions collected in Okinawa prefecture. These accessions could be distinguished genetically using three SSR markers, with the exception of accessions between ‘Uruma White’/‘Nago White’ and ‘Ohgimi Red 1’/‘Ohgimi Red 2’. Genetic diversity analyses suggested six groups, and grouping appeared to be based on differences in species. The obtained SSR markers and clarified genetic background information may facilitate future breeding of pitaya.