Bifidobacterium is one of the dominating bacterial genera in the honey bee gut, and they are the key degrader of diet polysaccharides for the host. Previous genomic analysis shows that they belong to separate phylogenetic clusters and exhibited different functional potentials in hemicellulose digestion. Here, three novel strains from the genus Bifidobacterium were isolated from the guts of the honey bee (Apis mellifera). Phylogenomic analysis showed that the isolates could be grouped into four phylogenetic clusters. The average nucleotide identity values between strains from different clusters are <95%, while strains in Cluster IV belong to the characterized species Bifidobacterium asteroides. Carbohydrate-active enzyme annotation confirmed that the metabolic capacity for carbohydrates varied between clusters of strains. Cells are Gram-positive rods; they grew both anaerobically and in a CO2-enriched atmosphere. All strains grew at a temperature range of 20-42 °C, with optimum growth at 35 °C. The pH range for growth was 5-9. Strains from different phylogenetic clusters varied in multiple phenotypic and chemotaxonomic characterizations. Thus, we propose three novel species Bifidobacterium apousia sp. nov. whose type strain is W8102T (=CGMCC 1.18893 T = JCM 34587 T), Bifidobacterium choladohabitans sp. nov., whose type strain is B14384H11T (=CGMCC 1.18892 T = JCM 34586 T), and Bifidobacterium polysaccharolyticum sp. nov. whose type strain is W8117T (=CGMCC 1.18894 T = JCM 34588 T).