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So far, nearly all studies concerning the mating frequency of bumblebees have been conducted with temperate species, showing that single mating seems to be the predominant pattern in bumblebees. Studies involving tropical species, however, are still scarce. Here, we determined the mating frequency of queens of the tropical bumblebee species, Bombus wilmattae by using microsatellite genotyping based on a sample of nine colonies from Chiapas, Southern Mexico. A total of 204 workers were genotyped with microsatellite markers to infer the queen genotype and the number of males with which each queen had mated. Two of the nine queens were doubly mated and seven singly mated. In the colonies with the double-mated queens, the distribution of the patrilines was not even, resulting in effective mating frequencies of 1.34 and 1.70, respectively, and an average relatedness of g = 0.58 ± 0.06. Inglés