Recently, Mycobacterium infection has been observed in cultured yellowtail Seriola quinqueradiata in Japan but not studied in detail. Diseased fish were lethargic, anorexic and emaciated, and showed hemorrhagic cutaneous ulceration and ascites. The necropsy and histopathological features showed that disseminated necrosis and numerous white nodules were found in the kidney, spleen, liver and heart. Numerous acid-fast bacteria were detected in the above tissues and granulomas. Myositis, hepatitis, splenitis and nephritis due to granulomas and gill inflammation were histologically observed. Almost all granulomas were classified into soft tubercle-type. All bacteria isolated from the diseased fish were Gram-positive, acid-fast, rod and non-motile. As a result, they were classified into the genus Mycobacterium. The isolates were identified as Mycobacterium marinum on the basis of biological and biochemical characteristics and the analysis of a partial 16S rRNA gene sequence. An experimental infection test showed that a representative isolate had pathogenicity to yellowtail with disease signs similar to those of naturally affected fish. This is the first report on M. marinum infection in cultured yellowtail.