The western honeybee, Apis mellifera, depends heavily on proper nutrition during overwintering, when colonies survive entirely on stored food resources. Many beekeepers replace natural honey with sugar-based feeds, but the effects of these diets on the honeybee gut microbiome are still not fully understood. This study investigated how different overwintering diets, including honey, sugar syrup, and a mixed honey-sugar diet, affect the gut microbiome of worker honeybees, with a special focus on the ileum, an important but understudied gut region. Worker bees were collected from three dietary treatment groups in Västra Götaland, Sweden, during early spring 2025. Ileal samples were dissected under sterile conditions, and full-length 16S rRNA gene sequencing was performed using Oxford Nanopore MinION technology to improve bacterial identification. Microbial diversity and bacterial composition were analyzed using taxonomic and statistical approaches. The results showed that core bacterial genera such as Gilliamella, Lactobacillus, and Snodgrassella were present in all dietary groups, indicating that the honeybee gut microbiome remained generally stable during overwintering. Bees fed with honey showed slightly higher microbial diversity compared with bees fed sugar or mixed diets. Some differences in the abundance of certain bacterial taxa were also observed between dietary treatments. Comparisons between the ileum and rectum revealed similar dominant bacterial groups, although the rectum contained greater bacterial abundance and variation. Overall, the study suggests that the overwintering diet can influence the balance and diversity of gut bacteria, while the core microbiome of honeybees remains relatively resilient under different feeding conditions.