There are limited reports of the genetic characterization of Toxoplasma gondii infecting captive macropods in North America. A novel genotype, ToxoDB PCR-RFLP genotype 263, was reported from six wallabies at a zoological facility in Virginia, USA, prompting an investigation into the genotypes from T. gondii strains infecting macropods at a zoological park in Florida, USA. Cardiac muscle and/or lung samples from an agile wallaby (Macropus agilis, n = 1), red kangaroos (Macropus rufus, n = 8), red-necked wallaby (Macropus rufogriseus, n = 1), and a tammar wallaby (Macropus eugenii, n = 1) that died between 2014 and 2018 were collected. All 11 cases were confirmed to have died from systemic toxoplasmosis by histopathology and immunohistochemical staining. Multilocus PCR-RFLP genotyping of T. gondii was performed directly on tissue samples or on parasites isolated from myocardium by mouse bioassay. Two cases of toxoplasmosis were identified as the reported novel genotype, ToxoDB PCR-RFLP genotype 263, but no common source of exposure could be identified. Five cases were identified as genotype 2 (type III strain, haplogroup 3), and four cases were identified as genotype 216, which has been previously reported in North American wildlife. There were no overt differences in lesion severity or distribution related to genotype. These results suggest that the premise was contaminated with at least three genotypes of T. gondii causing systemic toxoplasmosis in macropods. The largest cluster of fatal toxoplasmosis in macropods in the study period occurred following severe rainfall flooding of the exhibit, suggesting the transmission of T. gondii by water and pointing out the importance of this transmission mechanism. In summary, our study revealed three T. gondii outbreaks that caused significant loss of macropods within 5 yr in a zoological facility in Florida. More studies are needed to understand transmission and prevention of toxoplasmosis in sensitive zoo animals.