Honey bees are important pollinators in both agriculture and ecosystems, and their health is essential for sustainable human development. Although only two bacteria, Paenibacillus larvae and Melissococcus plutonius, have been identified as bacterial pathogens in honey bee brood for over 100 years, we found three additional Paenibacillus strains (Paenibacillus sp. J27TS7, Paenibacillus azoreducens J34TS1, and Paenibacillus melissococcoides J46TS7) in honey that harmed honey bee brood development. In particular, Paenibacillus sp. J27TS7 was highly virulent in bee larvae (the median lethal dose [LD50] = 12.7 spores/larva) and was comparable to P. larvae (LD50 = 2.3-11.5 spores/larva). Paenibacillus azoreducens J34TS1 showed the second-highest virulence (LD50 = 45.9 spores/larva), and P. melissococcoides J46TS7 was the least virulent (LD50 = 469.0 spores/larva). However, P. melissococcoides was most frequently detected in Japanese honey among the three species, with the highest concentration being 1.8 × 106 spores/mL honey, suggesting its wide distribution in Japanese apiaries. The novel pathogenic Paenibacillus species were categorized into the fast killer (Paenibacillus sp. J27TS7), medium-fast killer (P. melissococcoides), and slow killer (P. azoreducens) like P. larvae strains in terms of the time to kill infected brood; however, histopathological and genome analyses indicated that their pathogenic mechanisms were different from those of P. larvae strains. Moreover, P. melissococcoides showed differences in virulence depending on the lineage of the strain. These findings represent the first discovery of honey bee brood pathogens in more than 100 years and indicate the need to look beyond known pathogens for a comprehensive understanding of honey bee diseases.