Infection with multiple drug-resistant strains of Vibrio anguillarum was observed in ayu (Plecoglossus altivelis) farms from 1981 to 1983. The strains collected from various areas of Japan showed resistance to various combinations of nine drugs : chloramphenicol (CP), tetracycline (TC), streptomycin (SM), ampicillin (ABPC), colistin, nalidixic acid, furazolidone, sulfamonomethoxine (SA), and trimethoprim (TMP). The detected R plasmids showed resistance to CP, TC, SM, ABPC, SA, and/or TMP. The digestion patterns of the R plasmids were identical. These R plasmids also showed high homology with the R plasmids detected in 1980 as determined by the Southern blot hybridization method. In other words, drug resistant strains of V. anguillarum carrying R plasmids with the same DNA structure have been distributed in ayu farms since 1980 in Japan.