Comamonas thiooxidans (strain S23(T)) capable of oxidizing thiosulfate under a mixotrophic growth condition was isolated from a sulfur spring. DNA-DNA homology study showed 55% similarity with Comamonas testosteroni KCTC2990(T) and 52% with Comamonas composti LMG24008(T), the nearest phylogenetic relative (16S rRNA sequence similarity < 97%). Comparative genomic fingerprinting by using ERIC and Rep-PCR further delineated species identity of the strain S23(T) for which Comamonas thiooxidans sp. nov. is proposed. In addition, thiosulfate oxidation potential of the strain S23(T) was compared with Comamonas testosteroni and Comamonas composti.