Cryptosporidium is a human pathogen which can be transmitted through contaminated water and therefore is of particular interest to the drinking water industry. It has become generally understood that not all of the known species and genotypes of Cryptosporidium are pathogenic to humans and that they display a certain degree of host specificity. Understanding the distribution of Cryptosporidium species in raw water would provide an improvement to existing regulatory monitoring frameworks aimed at protecting public health from risks associated with drinking water. USEPA Method 1623 is an accepted method for monitoring Cryptosporidium occurrence, and in many countries is a regulation for the water industry. However, this method provides no information on the species/genotypes present, and likely overestimates human health risk. This thesis describes the development and validation of a molecular tool applied to Method 1623 processed microscope slides for effective molecular characterization of Cryptosporidium oocysts in water. These molecular tools were applied to 601 Cryptosporidium positive microscope slides from four agricultural watersheds located in geographically diverse regions of Canada. Identification of Cryptosporidium species/genotypes was successful from 422 of the samples analyzed. Species considered to be of high risk to humans through waterborne routes of transmission (C. hominis and C. parvum) were present in 3.8% of Cryptosporidium microscope positive samples, resulting in an overall frequency of 1.2% (based on the total number of water samples analyzed across all watersheds [n=1296]), suggesting a relatively low risk to human health. Wildlife was the most dominant source of Cryptosporidium contamination in the watershed followed by agriculture. Species originating from wildlife represented a low risk to human health. Spatial and temporal variation was also observed across the watersheds for Cryptosporidium occurrence, host source attribution and individual species or genotypes. Ultimately, these approaches can be used to characterize sources of fecal pollution in the environment and aid in the development of mitigation strategies focused on reducing the burden of Cryptosporidium in drinking water sources in an effort to protect public health.