A visceral mycosis occurred in ayu Plecoglossus altivelis larvae at Yamanashi Prefectural Fisheries Technology Center, Japan, in 2007 and 2008. Cumulative mortalities due to the disease were 19-33%. Most diseased fish were characterized by the opaque abdomen. Abundant non-septate hyphae with a width of approximately 5 pm were observed in the opaque areas. Fungi isolated from diseased fish were all identified as Pythium flevoense based on the morphological characteristics and sequence analysis of the 5.8S rDNA and adjacent ITS regions. Histopathological examinations showed that non-septate hyphae were present in the airbladder, kidney, intestine, pancreas, spleen, abdominal cavity, musculature and spinal cord. Heavy hyphal propagation in the airbladder and rhexis of the organ suggested that accidental ingestion of P flevoense into the airbladder was the prime cause of this disease.