Honey bee gut microbiota play specific roles in promoting host growth and physiology by regulating the immune system, behavior, metabolism, and neurological processes. While the gut microbiota of honey bee queens, workers, and larvae has been extensively studied, less is known about the composition of gut microbiota in the winter worker bees. This study investigates the dynamics of the gut microbiota in overwintering adult worker bees, focusing on two commercial bee strains: Bolton™ bees and Mann Lake™ bees. These Apis mellifera strains were investigated under different storage conditions (indoor storage at 6 °C and outdoor storage in natural conditions) during the winter months (October, November, and December). Utilizing 16S rRNA gene amplicon sequencing, we characterized the microbial composition of the whole gut. We observed the Lactobacilli dominated in all the overwintering honey bee guts with a significantly higher abundance of unclassified Lactobacillus species in November, while Lactobacillus apis showed significantly higher abundance in October. Bolton bees exhibited significantly higher abundance levels of Bartonella (denoted as uncultured) and Bifidobacterium, along with an unexpected presence of Wolbachia. In contrast, Mann Lake bees demonstrated an increased abundance of Commensalibacter. Our results suggest that Shannon diversity is influenced by the month rather than by the bee strain or storage conditions. We also found significant differences in Bray Curtis diversity index by month. Overall, taxonomical abundance was not affected by whether the hives were stored outside or in constant temperature indoor storage. However, various bacterial species showed differences in abundance across different months, with slight variations observed between bee strains. Given the potential benefits of the honey bee gut microbiome for health and nutrition, our data suggests that the genus Lactobacillus may play a significant role in bee health during winter and overwintering storage.