Utilization of zebrafish, Danio rerio, has steadily increased and its applications have expanded into numerous fields of science. Applying elevated temperatures (32°C to 37°C) to this organism has allowed researchers to conduct climate change, human cancer and infectious disease studies. Though zebrafish can be acclimated from the standard 28°C to higher temperatures, most studies use larval fish so consequences of increased temperature have not been thoroughly characterized in adult fish. Moreover, these studies neglect to consider the effect of temperature on common pathogens of fish and how susceptibility to infections may be affected. Mycobacterium chelonae is ubiquitous in the environment and causes chronic infections in zebrafish colonies. Mycobacteriosis in fish populations may be asymptomatic but may also be a major source of non-protocol induced variation that could affect results of any study that involves zebrafish. The goal of my study is to characterize possible differences in M. chelonae growth at 28°C compared to 35°C and to describe infectivity and susceptibility of zebrafish to the bacteria at the two temperatures. Several M. chelonae and related mycobacteria isolates demonstrated significantly increased colony growth on agar plates at 35°C compared to 28°C. For M. chelonae H1E2, colonies at higher temperatures were 2-fold larger, though they experienced more cell death. When the bacteria was grown in a broth medium, growth rates appeared similar at the two temperatures. In the first of two in vivo experiment, fish injected with M. chelonae had significantly higher bacterial loads at 28°C than 35°C when analyzing bacterial abundance between populations (P<0.002, Wilcoxon test). However, there were no significant differences in infection intensity (P>0.178, Wilcoxon test). Histology showed evidence of mycobacteriosis in all groups of fish, including a few control fish. One control fish had clear signs of mycobacteriosis that was not identified as M. chelonae by ddPCR. Fish from all groups also had acid-fast bacteria in the gut lumen. The second in vivo experiment showed significantly higher bacterial loads in fish held at 28°C in comparison to 35°C for both abundance (P<0.003, Wilcoxon test) and intensity (P<0.02, Wilcoxon test). Based on the evidence from the culture-based growth experiments, histopathology and molecular analysis, I conclude that it is possible to establish infections at higher temperatures in zebrafish, the risk of developing a M. chelonae infection and disease decreases as temperature increases.