This prospective multicenter surveillance study was designed to provide antimicrobial susceptibility profiles of clinical anaerobic bacteria with genetic species identification in Japan. In 2014, a total of 526 non-duplicate clinical anaerobic isolates were collected from 11 acute-care hospitals in the Kyoto and Shiga regions of Japan. Genetic identification was performed using 16S rRNA sequencing. Minimum inhibitory concentrations were determined in the central laboratory and were interpreted using the CLSI criteria. Genetic analysis provided species-level identification for 496 isolates (83 species in 40 genera) and genus-level identification for 21 isolates (13 genera). Among these 517 isolates, the most frequent anaerobes were Bacteroides spp. (n = 207), Prevotella spp. (n = 43), Clostridium spp. (n = 40), and Peptoniphilus spp. (n = 40). B. fragilis was the most common species (n = 107) and showed 91.6%-97.2% susceptibility to β-lactam/β-lactamase inhibitor combinations (BLBLIs; ampicillin-sulbactam, amoxicillin-clavulanate, and piperacillin-tazobactam) and carbapenems (imipenem and meropenem) as well as 100% susceptibility to metronidazole. Gram-negative anaerobes were highly susceptible to metronidazole (99.0%) followed by BLBLIs and carbapenems (>90% each). BLBLIs or carbapenems also retained activity against Gram-positive anaerobes (99.5%-100%) except Clostridioides difficile. All isolates were susceptible to combinations of metronidazole with BLBLIs or carbapenems. Thus, BLBLIs or carbapenems are first choices for empirical therapy of anaerobic infections in Japan, and these antimicrobials in combination with metronidazole should be reserved for very severe infections and targeted therapy.