In honeybees, the mechanism of sex determination depends on genetic variation at the complementary sex determiner ( CSD ) locus, which has a large allelic diversity. In this study, we examined the population genetic structure and genetic diversity within the highly variable region ( HVR ) of CSD in five Apis mellifera subspecies, in addition to Buckfast and unknown mixed ancestry bees. We sequenced CSD in 329 drones, 146 from Algeria ( A. m. intermissa and A. m. sahariensis subspecies) and 183 from Europe ( A. m. ligustica , A. m. carnica , A. m. mellifera subspecies, Buckfast samples, and individuals of unknown mixed ancestry). A total of 119 nucleotide haplotypes were detected. These corresponded to 119 protein haplotypes, of which 81 were new. The analysis of these haplotypes showed that HVR diversity levels were comparable with those in other populations of honeybee worldwide. Paradoxically, this high level of diversity at the locus did not allow for a separation of the samples according to their subspecies origin, which suggested either an evolutionary convergence or a conservation of alleles across subspecies, and an absence of genetic drift. Our results can be used to provide more information about the CSD diversity to include in breeding programs of honeybee populations.