Genetic differences between trilinear and dilinear forms of a tongue sole, Cynoglossus interruptus, were examined by use of isozymes. Unequivocal differences were detected between the two forms, including complete replacement of alleles at the FBALD-2*, MDH-3*, PROT-1*, and SOD-1* loci, almost complete replacement at the AAT-3*, AH-1*, AH-2*, EST-3*, G3PDH-3*, GPI-2*, IDHP-1*, and MDH-1* loci, and extreme differences in allelic frequencies at the GPI-1* and PGDH* loci. The genetic distances (D values) between the two forms were 0.4207–0.4353, figures predicatively significant at the specific level. The considerable genetic differences strongly suggested that the two forms represent distinct species.