The common brushtail possum (Trichosurus vulpecula) is one of the most abundant native marsupials in urban Australia, having successfully adapted to utilize anthropogenic resources. The habituation of possums to food and shelter available in human settlements has facilitated interaction with people, pets, and zoo animals, increasing the potential for transmission of zoonotic Cryptosporidium pathogens. This study sought to examine the identity and prevalence of Cryptosporidium species occurring in possums adapted to urban settings compared to possums inhabiting remote woodlands far from urban areas and to characterize the health of the host in response to oocyst shedding. Findings indicated that both populations were shedding oocysts of the same genotype (brushtail possum 1 [BTP1]) that were genetically and morphologically distinct from zoonotic species and genotypes and most closely related to Cryptosporidium species from marsupials. The urban population was shedding an additional five Cryptosporidium isolates that were genetically distinct from BTP1 and formed a sister clade with Cryptosporidium parvum and Cryptosporidium hominis. Possums that were shedding oocysts showed no evidence of pathogenic changes, including elevated levels of white blood cells, diminished body condition (body mass divided by skeletal body length), or reduced nutritional state, suggesting a stable host-parasite relationship typical of Cryptosporidium species that are adapted to the host. Overall, Cryptosporidium occurred with a higher prevalence in possums from urban habitat (11.3%) than in possums from woodland habitat (5.6%); however, the host-specific nature of the genotypes may limit spillover infection in the urban setting. This study determined that the coexistence of possums with sympatric populations of humans, pets, and zoo animals in the urban Australian environment is unlikely to present a threat to public health safety.Cryptosporidium is a genus of protozoan parasite that causes enteric disease in a range of vertebrates with potentially fatal consequences for young or immunocompromised individuals (17). While most species within the Cryptosporidium genus exhibit strong adaptation to the host (55), eight Cryptosporidium species and genotypes from animals (C. parvum, C. meleagridis, C. suis, C. baileyi, C. canis, C. felis, C. muris, and the cervine genotype) have zoonotic capacity and present a public health risk (8,33,41). To assess the threat of spillover infection, a growing number of studies have characterized Cryptosporidium species circulating in wildlife populations. To date, most studies have focused on rodents (5, 9, 13) and a range of animals that, by inhabiting natural watersheds, pose a risk of contaminating human drinking water supplies (2,22,31,34,36,58). In contrast, few studies have investigated the epidemiology of Cryptosporidium in wildlife that live directly at the interface of urban settlements, despite their coexistence with high densities of humans and domestic pets, a situation that has proven conducive ...