Neoseiulus womersleyi (Schicha) (Acari: Phytoseiidae) is one of the most important predators of spider mites in Japan. Various characteristics have been studied in this species. However, because there is a lack of genetic markers, genetic diversity within and among populations has not been well elucidated. Microsatellites, short stretches of tandem-repeated 1- to 5- nucleotide sequences, are ubiquitously present in eukaryotic genomes and are highly polymorphic. Their high polymorphism makes them suitable markers for studying intra-specific variation. In this study, we developed microsatellite markers in N. womersleyi, and then examined genetic diversity in their populations. Microsatellite enriched genomic DNA library was contracted and sequenced from a single female adult. Of the 40 plasmids sequenced, 31 plasmids showed microsatellite sequences and 24 plasmids were unique. Finally, we could design primers on three loci. When tested their diversity on one wild and two laboratory populations, five to 18 alleles were detected. The wild population showed highest genetic diversity, and this divergence decreased in rearing populations. To investigate the effects of different rearing conditions, genetic diversity in two rearing populations, which were different in population size, were compared with those in the original wild populations. The allelic richness and gene diversity were not significantly different between wild and large-size populations, while the values were significantly decreased in small-size populations. Thus, 40 to 60 females per generation was sufficient to conserve the genetic diversity in N. womersleyi populations during laboratory rearing. In conclusion, the microsatellite markers developed were useful to evaluate genetic diversity in wild and laboratory populations of N. womersleyi.