Three tributaries of the Grand River watershed (Ontario, Canada), each representing different watershed types (urban, agricultural/rural, and mixed land-use) were examined to understand the spatial, temporal, and host-source distribution of the waterborne pathogen, Cryptosporidium. Cryptosporidium was frequently found throughout the study (73%, 65/89) with occurrence and concentrations observed to be similar among the varying watershed types. However, applying advanced genotyping techniques, marked differences in dominant host sources could be observed in each watershed. The agricultural/rural and mixed land-use watersheds were dominated by genotypes typically associated with cattle (i.e., C. andersoni), while the urban watershed had the highest diversity of Cryptosporidium genotypes with a variety of wildlife as the common source of contamination (e.g., muskrat and cervine genotypes). A similar seasonal trend observed in the urban, agricultural, and mixed land-use watershed suggests that factors beyond specific land use activities (e.g. autumn manure spreading) may influence the timing and concentration of Cryptosporidium in these streams. Corresponding genotyping results provided additional insight into source inputs during these seasonal peaks, indicating that wildlife may be important seasonal contributors to Cryptosporidium contamination in these streams. Despite the abundance of Cryptosporidium in these watersheds, most of the genotypes observed were of limited human health importance. This study provides evidence regarding the significance of including genotyping results into studies examining waterborne Cryptosporidium. Using this technique can provide a greater understanding of the risk to the population using water sources, as well as provide insight into the probable sources and timing of contamination. This ancillary information can contribute to implementation of targeted management strategies to further protect sources of drinking water and recreation areas.