The repeated evolution of extreme polyandry in advanced social insects is exceptional and its explanation has attracted significant attention. However, most reported estimates of the number of matings are derived from limited sampling. Temporal and geographic variation in mating behavior of social insects has not been sufficiently studied. Worker offspring of 18 Eastern Honey Bee (Apis cerana Fabr.) queens from three populations across Thailand were genotyped at five microsatellite markers to test for population differences of mating behavior across three different ecosystems. The number of matings decreased from a northern, more seasonal environment to a southern tropical population and was lowest in a tropical island population. Our study confirms earlier findings that social insect mating behavior shows biogeographic variation and highlights that data from several populations are needed for reliable species-specific estimates of the number of matings. Detailed studies of populations that show significant differentiation in the number of matings may be able to discriminate effectively among the different hypotheses that have been proposed to explain the evolution of polyandry in honey bees and other advanced social insects.