A retrospective analysis of archived tissue blocks has revealed that mycobacteriosis was apparent in Chesapeake Bay striped bass as early as 1984. Of 37 cases available from the years 1975 to 1985, 2 fish were found positive based on histopathology and genus-specific PCR. Multi-gene sequencing places the bacteria from the 2 positive cases (1984 and 1985) within the Mycobacterium tuberculosis clade with closest resemblance to the recently described fish pathogen M. pseudoshottsii. Our data confirms that mycobacteriosis is not a new disease of Chesapeake Bay striped bass and underscores the value of archived tissues in epidemiological examinations.