A Lancefield group C streptococcal (GCS) infection caused by Streptococcus dysgalactiae that is characterized by severe necrotic lesions of the caudal peduncle has been an increasing cause of mortality in farmed fish such as amberjack, Seriola dumerili, and yellowtail, Seriola quinqueradiata, in the southern part of Kyushu, Japan. In this study, enzymatic profiles of GCS strains from fish and mammals were investigated using the API ZYM system, and genotypic characterization of GCS strains was performed using biased sinusoidal field gel electrophoresis (BSFGE). The partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish and mammals was also compared. The API ZYM test indicated that it is difficult to differentiate isolates of S. dysgalactiae from fish and animals based on enzymological variations. In the BSFGE analysis, the macrorestriction profiles, which were obtained using SmaI or ApaI as a restriction enzyme, revealed variations between the fish and animal isolates. The partial sequence of the 16S-23S rDNA intergenic spacer region of all the tested fish isolates differed from all mammalian isolates in one or two nucleotides. The possibility of a clonal expansion of S. dysgalactiae strains in farmed fish was also suggested by the BSFGE profiles of fish isolates.