Investigation into recent declines in striped bass health in the Chesapeake Bay in Maryland resulted in the isolation of a putative new species of Mycobacterium. This isolate was obtained from fish showing skin ulcers and internal granulomas in various organs. The isolate was slow growing at 28°C; was nonchromogenic; showed no activities of nitrate reduction, catalase activity, Tween 80 hydrolysis, tellurite reduction, or arylsulfatase reduction; grew best at low salt concentrations; and was urease and pyrazinamidase positive. By PCR a unique insertional sequence was identified which matched nothing in any database. Analysis of the nearly complete 16S rRNA gene sequence also indicated a unique sequence which had 87.7% sequence homology to Mycobacterium ulcerans, 87.6% homology to Mycobacterium tuberculosis, and 85.9% homology to Mycobacterium marinum. Phylogenetic analysis placed the organism close to the tuberculosis complex. These data support the conclusion that the isolate probably represents a new mycobacterial species.