Males of some eusocial hymenopterans live in sheltered hives where they are raised by sister workers until they are ready to mate. Large amounts of colony resources are invested in the care and nurturing of males, as they provide no contributions to colony maintenance apart from reproduction. Colonies of the honey bee, Apis mellifera L., have one queen, thousands of female workers, and a few thousand seasonal males (drones) that are reared only during the reproductive season when colony resources are plentiful. We examined the viability of spermatozoa in sexually mature drones from eight apiaries in three counties in Central Texas during the summers of 2013 and 2014. We sampled 1,622 drones from two counties in 2013, and 556 drones from three counties in 2014. Using dual fluorescent flow cytometry, viability of drone spermatozoa was measured as the proportion of total spermatozoa that was viable. The average spermatozoa viability was 46.2% in 2013 and 67.0% in 2014. We found significant variation in spermatozoa viability across apiaries, with viability in Apiary 5 significantly lower and viability in Apiary 11 significantly greater than viability in the other apiaries sampled in 2013. Likewise, males in apiaries 6 and 7 had significantly lower average viability of spermatozoa compared with other apiaries analyzed in 2014. However, the changes were not consistent across apiaries during the 2 years sampled. Our results suggested that other factors such as exposure to insecticides or seasonal availability of forage might be more important than inter-colonial genetic differences in fertility of honey bee drones during the reproductive season.