The Varroa destructor is an ectoparasitic mite and the most serious biotic threat to honey bee and apiculture worldwide. Genetic types of V. destructor determine its ability of transmission and pathogenicity. Population genetics tools could supply useful information for comprehensive management of the mite. In this study, transcriptome information of V. destructor was analyzed for mining more polymorphic microsatellite markers for the population genetics investigation with further experimental verified. A total of 83,711 unigenes were assembled with the N50 length of 1,826 bp and the GC content of 40.03%. A total of 27,775 potential microsatellite loci were identified in 18,563 unigenes. The di-nucleotide and mono-nucleotide were most abundant repeat motifs and the most dominant di-nucleotides and mono-nucleotide repeat motifs were A/T and AT/TA. Forty-two of sixty randomly selected microsatellite loci were successfully amplified. Six of them were confirmed to be polymorphic in five V. destructor geographical populations. Our result showed transcriptomic data provide valuable resources for molecular marker development, and these novel microsatellite loci would be valuable in facilitating population genetic and evolutionary of V. destructor and related species.