The phenotypic and genetic characterizations of 58 isolates of the fish pathogen Nocardia seriolae, from amberjack, Seriolae dumerili, yellowtail, Seriola quinqueradiata, Japanese flounder, Paralichthys olivaceus, and chub mackerel, Scomber japonicus, in Japan from 1970-2005, were examined to investigate the epidemiological relationship between isolates. The phenotypic and genetic characterizations were determined by alpha-glucosidase activity and biased sinusoidal field gel electrophoresis (BSFGE) analysis, respectively. There was no alpha-glucosidase activity in strains isolated from 2000-05 (n = 50) with a few exceptions (n = 3), while all strains isolated from 1970-90 (n = 8) were positive. In BSFGE analysis, digestions with restriction enzymes Xba I and Ase I produced 15 and 16 restriction patterns, respectively. All restriction patterns obtained from 50 strains isolated during 2000-05 were unrelated to those obtained from eight strains isolated during 1970-90, with the exception of two strains isolated during recent outbreaks. Based on the phenotypic and genetic characterizations, recent outbreaks of nocardiosis in Japan are suggested to be epidemiologically unrelated to earlier outbreaks in Japan. Although a low genetic relationship was observed in the restriction pattern between recent and earlier isolates, identity was confirmed between these groups of isolates because five representative strains showed 99.9% homology with N. seriolae ATCC43993(T) in the 16S rRNA sequence.