The storage of spermatozoa and keeping alive for years in the spermatheca by the honey bee queen is a phenomenon allowing her to fertilize eggs throughout her life. In this study, the queens that were 2-week, 1-year, and 2-year-old were analyzed to determine the viability of spermatozoa in their spermathecae. The sister queens reared by the grafting method were instrumentally inseminated with 8 μl fresh semen when they became 6 days old. One week after instrumental insemination, one batch of queens (2-week-old) was dissected for spermatozoa viability test. Another set of queens was introduced into production colonies in Langstroth hives after the onset of oviposition in the mating nuclei. The queens were maintained in production colonies for one year and two years until the dissection process for spermatozoa viability test. The viability of spermatozoa was measured by the dual staining method. We determined that the mean viability of spermatozoa in 2-week, 1-year and 2-year-old queens were 97.3%, 91.1% and 88.1%, respectively. The viability of spermatozoa in queens decreased with age, and the differences between the viability means were significant. However, we did not detect a steep decline in the viability of spermatozoa in queens (6% in one year and 9% in two years) in a wide range of timescale in contrast to previous reports. Furthermore, we found high viability of spermatozoa in the spermathecae of queens at the start of their lives and one- and two-years of age.