We estimated queen mating frequency, genetic relatedness between workers and worker reproduction in the hornets Vespa analis and Vespa simillima using microsatellite DNA genotyping. The 20 V. analis colonies studied each contained a queen inseminated by a single male. Of the 15 V. simillima colonies studied, nine had a queen inseminated by a single male, four had a queen inseminated by two males, and two had a queen inseminated by three males. The estimated effective number of matings was 1.33 ± 0.74 (mean ± SD), with 75–85% of the offspring of the six multiply mated queens sired by single males. The values for genetic relatedness between the workers of V. analis and V. simillima were 0.739 ± 0.004 and 0.698 ± 0.013 (mean ± SD), respectively. We conclude that V. analis and V. simillima colonies are genetically monogynous and monandrous. When high relatedness between the workers occurs within colonies, kin selection theory predicts a potential conflict between queens and workers over male production. To determine whether males were derived from queens or workers, males from V. analis and V. simillima colonies were genotyped at four microsatellite loci and the level of ovary activation in workers was determined. None of the 787 V. analis workers and only 15 of 3520 V. simillima workers had developed ovaries. Furthermore, the genotyping identified no worker-produced males in any colony. The presence of reproductive workers correlated positively with the number of workers within the colony. These results suggest that eusocial colonies with an annual life cycle tend to break down socially when they become large and are close to dying.