Fecal contamination of surface waters is one the major sources of waterborne pathogens and consequently, is an important concern for public health. For reliable fecal source tracking (FST) monitoring, there is a need for a multi-marker toolbox as no single all-encompassing method currently exists. Mitochondrial DNA (mtDNA) as a source tracking marker has emerged as a promising animal-specific marker. However, very few comprehensive field studies were done on the occurrence of this marker in surface waters. In this report, water samples were obtained from 82 sites in different watersheds over a six year period. The samples were analyzed for the presence of human, bovine and porcine mtDNA by endpoint nested PCR, along with the human-specific Bacteroidales HF183 marker. These sites represented a mix of areas with different anthropogenic activities, natural, urban and agricultural. The occurrences of mitoHu (human), mitoBo (bovine), mitoPo (porcine) and HF183 specific PCR amplifications from the samples were 46%, 23%, 6% and 50%, respectively. The occurrence of mitoHu and HF183 was high in all environment types with higher occurrence in the natural and urban areas, whereas the occurrence of mitoBo was higher in agricultural areas. FST marker concentrations were measured by real-time PCR for samples positive for these markers. The concentration of the mitoHu markers was one order of magnitude lower than HF183. There was co-linearity between the concentrations of the mitoHu and HF183 markers. Co-linearity was also observed between HF183 concentration and fecal coliform levels. Such a relationship was not observed between the mitoHu concentration and fecal coliform levels. In summary, our results showed a high incidence of human fecal pollution throughout the environment while demonstrating the potential of mtDNA as suitable FST markers.