The objective of this study was to test six diluents, three cryoprotectants, and five semen:diluent ratios as a means to improve post-thaw viability of cryopreserved honey bee ('Apis mellifera' L.) semen for instrumental insemination. In addition, differences in sperm freezing tolerance among strains of honey bees were tested. Specific protocols were designed to control freezing and thawing rates. Spermatozoa motility was assessed visually, while viability was assessed using SYBR-14 and propidium iodide. There were no significant differences among strains for post-thaw spermatozoa viability. Semen collected at high dilution ratios, using diluent 4 in combination with cryoprotectant DMSO, provided significantly higher post-thaw viability than all other combinations tested (68.3 ± 5.4%). These new semen dilution and freezing methods improve post-thaw viability of spermatozoa to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.