Surface waters used as sources of drinking water can be at risk for pathogen contamination from non-point source pollution. Non-point source pollution is a problem because it can be difficult to detect and quantify. Source water protection is a tool to protect public health by maintaining and improving source water quality. The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR), which aims to improve the control of Cryptosporidium, recognizes watershed protection programs as part of both Cryptosporidium and virus removal requirements. Microbial source tracking (MST) is a tool that is currently being developed to aid in source water protection. The research project utilized an integrated approach to differentiating sources of microbial contamination to the West Boylston Brook (WBB) subwatershed, a tributary to the Wachusett Reservoir. The Wachusett Reservoir is part of the water supply for the Boston (MA) Metropolitan region. Since the Boston supply is unfiltered, a proactive approach has been implemented by the Massachusetts Department of Conservation of Recreation (DCR) to identify microbial contamination at the source. The WBB sub watershed was ranked most impacted in the Wachusett Reservoir as measured by fecal coliform levels, which were periodically high at the confluence with the reservoir, Gate 25 sampling location. The integrated approach used in this investigation included microbial source tracking applications, traditional microbial monitoring, land use analysis, and water chemistry monitoring. Land use analysis involved use of various maps and sanitary survey data to identify potential sources of microbial contamination, so that an effective sampling scheme could be developed. Residential and commercial developments, a dairy operation, and wildlife activity were identified as being potential sources of microbial contamination. Water samples were collected seasonally and under various precipitation conditions at selected sampling locations. The MST methods implemented at WBB included sorbitolfermenting Bifidobacteria (an indicator of human-related microbial contamination), Rhodococcus coprophilus (an indicator of grazing animal-related microbial contamination), and F+RNA coliphages (an indicator that can differentiate between human and non-human sources of microbial contamination). Water chemistry analyses included a suite of field and laboratory· measurements, while traditional. water quality indicators included the enumeration of both fecal coliforms and Escherichia coli. The results of the study indicated that the MST methods provided specific information on microbial input sources, when compared to the traditional water quality parameters alone. Sorbitol-fermenting Bifidobacteria and Rhodococcus coprophilus were both detected in samples where land use analysis would predict their presence. Overall, microbial source tracking, along with land use analysis, water chemistry monitoring, and traditional microbial monitoring, proved useful in differentiating sources of microbial contamination when combined with a hydrologically-based sampling design. The overall data analyses confirmed that a complete watershed control program should include water quality monitoring, MST monitoring, and seasonal sampling.