Abstract: Flavobacterium columnare, the causative agent of columnaris disease infects a wide range of freshwater fish throughout the world, with major symptoms being gill-rot and caudal rot. Gill-rot disease is also a major disease of freshwater fish in China; although its pathogen has been reported, disputes over its identity still exist. In order to understand the pathogen of gill-rot disease and the columnaris disease in China, bacterial pathogens were isolated from gill-rot diseased fish in fish farms in Hubei, Guangdong, Anhui, Sichuan Provinces and in suburbs of Beijing, China, respectively. The physiological and biochemical characters, growth in tobramycin-contained Shieh agar medium, and the formation of rizoids, as well as the ability in binding Congo red and in degrading gelatin and in production of chondroitinase were examined. These characteristics confirmed that the so-called gill-rot disease pathogen was actually the pathogen of columnaris disease, i.e. F. columnare. Furthermore, 16S rDNA sequences were cloned from 16 strains of F. columnare in the present study, and those downloaded from the GenBank database, a phylogenetic tree was built with the identification of three well-supported clades, corresponding clearly to the reported three genomovars of F. columnare. The first clade contains only three strains, including the most virulent strain G4 isolated in 1970s from grass carp, and two others from Japan and USA, respectively. The second and third clades contain many more strains, including those from same species of fish hosts, such as some cyprinid fish. But strains isolated from salmonids are clustered in the second clade, with the inclusion of a low-virulence strain G18 isolated in 1970s from grass carp in China. Strains isolated from the so-called Chinese perch, Siniperca chuatsi and Chinese sturgeon Acipenser sinensis in China are clustered in the third clade. The bacterium, Myxococcus piscicola Lu, Nie & Ko, 1975 named as the pathogen of gill-rot disease of grass carp Ctenopharyngodon idellus should be the synonym of F. columnare in terms of its similarity with F. columnare in above-mentioned characters and also in 16S rDNA. Further investigation should be carried out to examine if these strains in different clades differ in their virulence and then in pathogenicity.