Technical Abstract: Honey bee semen that was assayed for viability of spermatozoa using a dual fluorescent staining technique was very variable. Temperature of the buffer in which semen is collected had much less effect on viability than the way in which the semen was collected. Washing semen into the buffer did less harm than suctioning it up into a syringe. The highest viability levels were seen when semen was collected directly from seminal vesicles. The implications of these results for evaluation of semen in the development of germplasm preservation and for artificial insemination methods are discussed.