The parasitoid wasp Melittobia is an important insect for basic and applied biology. Specifically, their extremely female-biased sex ratios, which contrast to the prediction of pre-existing theories, are needed to be explained from the aspect of evolutionary biology. In this study, using next-generation sequencing, 20 microsatellite loci were developed and characterized for M. australica. The developed loci were used to analyze two populations, one from a mainland Japan and one from the Okinawa island region. Both populations showed a smaller observed heterozygosity than expected, and a high inbreeding coefficient. Deviations from Hardy-Weinberg equilibrium were recorded for the majority of the 20 loci, suggesting that both the populations are subdivided due to inbreeding as is expected by the reproductive biology in Melittobia. The sib-mating frequency in the two populations was calculated as 0.873 and 0.996, which is higher than the values estimated by the number of females observed in a host cocoon or cell, implying that closely related females lay eggs on a host. The microsatellite loci developed in this study can be used for more comprehensive analyses to identify genetic structure in natural populations for understanding their sex allocation behavior and for more generally evolutionary and population genetic studies.