Hybrids of Rhinogobius biwaensis and Rhinogobius sp. BF were identified in the ponds of Sento Imperial Palace, Kyoto City, Japan, from microsatellite and mitochondrial DNA markers. Mitochondrial DNA analysis indicated that five of seven Rhinogobius specimens had R. biwaensis haplotypes, the remaining specimens having those of Rhinogobius sp. BF. In DAPC and STRUCTURE analyses based on microsatellite data, all specimens examined showed intermediate status between R. biwaensis and Rhinogobius sp. BF, the two species being considered to have formed a hybrid swarm in the ponds because genetically pure individuals of either were not found. This is the first report of recent hybridization under field conditions in Rhinogobius fishes from Japan, confirmed by nuclear and mitochondrial DNA data. Hybrid Rhinogobius specimens from the ponds had 5–17 predorsal scales, an intermediate range between those of non-hybrid R. biwaensis (0–6) and Rhinogobius sp. BF (11–20). However, in appearance they were more similar to R. biwaensis than Rhinogobius sp. BF, having bluish-white distal coloring anteriorly on the first and second dorsal fins, a reduced reddish longitudinal band on the anal fin, and lacking yellowish dorsal and posterior margins on the caudal fin. However, they differed from R. biwaensis in having a narrower white posterior caudal fin margin. Further morphological differentiation of the hybrid specimens was not apparent.