Recent molecular evidence suggests that different species and/or genotypes of
Cryptosporidiumdisplay strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidiumspecies and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidiumparasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidiummuskrat genotype II, Cryptosporidiumcervine genotype, C. baileyi, C. parvum, Cryptosporidiummuskrat genotype I, the Cryptosporidiumfox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidiumparasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidiumparasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.