Mycobacteriosis was implicated in the deaths of eight Chinese gliding frogs (Rhacophorus dennysi) in a zoo population over a 3 yr period. Clinical signs included nonhealing skin lesions, cloacal prolapse, hind limb weakness, weight loss, and sudden death. Abnormalities on postmortem were proliferative or ulcerative skin lesions in four of eight, pneumonia in three of eight, and gall bladder empyema in two of eight cases. All eight clinical cases had multisystemic granulomas containing acid-fast bacilli. Tissues most commonly affected were lung (seven of eight), liver (six of eight), kidney (six of eight), spleen (five of eight), and heart (five of eight). The remaining eight clinically normal frogs in the population were euthanized: eight of eight had granulomatous lesions, with acid-fast bacilli in three of eight cases. A mycobacterial species was cultured from four of the clinical cases by the Public Health Ontario Laboratory and was initially misidentified as Mycobacterium tuberculosis complex by a commercial line-probe assay (GenoType Mycobacterium CM, Hain Lifesciences, Nehren, Germany). Further diagnostic testing using 16S rRNA gene sequencing ultimately identified the mycobacterial species as Mycobacterium marinum. The correct identification of mycobacterial species is essential in epidemiological investigations at zoological facilities, and in assessing health risks to staff and to other animals in the zoo population.