Preservation of honey bee (Apis mellifera L., Hymenoptera: Apidae) sperm, coupled with instrumental insemination, is an effective strategy to protect the species and their genetic diversity. Our overall objective is to develop a method of drone semen preservation; therefore, two experiments were conducted. Hypothesis 1 was that cryopreservation (–196 °C) of drone semen is more effective for long-term storage than at 16 °C. Our results show that after 1 yr of storage, frozen sperm viability was higher than at 16 °C, showing that cryopreservation is necessary to conserve semen. However, the cryoprotectant used for drone sperm freezing, dimethyl sulfoxide (DMSO), can harm the queen and reduce fertility after instrumental insemination. Hypothesis 2 was that centrifugation of cryopreserved semen to reduce DMSO prior to insemination optimize sperm quality. Our results indicate that centrifuging cryopreserved sperm to remove cryoprotectant does not affect queen survival, spermathecal sperm count, or sperm viability. Although these data do not indicate that centrifugation of frozen-thawed sperm improves queen health and fertility after instrumental insemination, we demonstrate that cryopreservation is achievable, and it is better for long-term sperm storage than above-freezing temperatures for duration of close to a year.