During routine screening of zebrafish at a research facility, histological changes consistent with mycobacteriosis were observed, prompting an investigation to determine the background prevalence and distribution of Mycobacterium species throughout the facility. Infection status was evaluated in 240 zebrafish representing 9 genetic lines, using histology, culture and PCR. Environmental sources were also tested for the presence of mycobacteria. Prevalence in zebrafish by culture and PCR was 10% (24/240), 21 of which were TU line fish. All isolates from fish were identified as M. chelonae by hsp65 DNA sequencing; subsequent DNA fingerprinting delineated 3 strains, designated H1E1 (1/24), H1E2 (22/24), and H1E3 (1/24). From external sources, tank or tubing surface biofilms were positive by culture (13/32) with multiple species and strains isolated including M. neoaurum, M. phocaicum, and identical strains of M. chelonae that were found in zebrafish: H1E1 (2/13) and H1E2 (8/13). Comparing diagnostic methods, acid-fast stained histological sections showed substantial agreement with plate culture and PCR for detection of mycobacteria in fish. Observation of granulomas in hematoxylin and eosin-stained sections was a less reliable predictor of mycobacteriosis, as uninfected females with egg-associated inflammation and hyperplasia were misdiagnosed. These data revealed background levels of mycobacteriosis in a healthy and well-managed facility. Infected populations were removed, although the apparent ability for M. chelonae to remain viable in environmental reservoirs may make it difficult to eradicate completely. This highlights the importance of an animal-health monitoring program and good husbandry practices to prevent disease in zebrafish research laboratories.