Yellowtail ascites virus (YTAV) is the causative agent of ascites and deformity in fish and causes serious losses to the fish-farming industry of yellowtail fry and fingerling Seriola quinqueradiata in Japan. In 2006, cultured yellowtail died from ascites in Kochi, Japan. We isolated and characterized a virus from the diseased fish. Based on the pathogenicity, culture characteristics, morphological features, RT-PCR results targeting VP2/NS region, phylogeny based on the VP1 amino acid sequence, and immunochemical reactivity of structural proteins, the virus isolate was identified as YTAV (designated as YTAV-06). YTAV-06 was a more virulent isolate than YTAV Y-6, isolated originally from yellowtail with ascites. To our knowledge, this is the first report describing that YTAV isolates may vary in their virulence.