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Santa Ana River in Southern California and its tributaries drain the largest volume of water among all rivers in Southern California. Most of the surface flow from the river is diverted to aquifers that supply water to approximately 2 million people. Chino Creek, a tributary of Santa Ana River (SAR), is an impaired water body due to pathogens and nutrients from agricultural activities, non-point source runoff, and population growth. This monitoring study was conducted to evaluate the diversity of the microbial populations during wet- and dry-weather conditions. Bacterial concentrations in surface waters from a number of locations along Chino Creek and an open-space site were determined using several different analytical methods. Most Probable Number (MPN) was used to monitor the concentration of viable indicator bacteria. A portion of the 16S rDNA genes was amplified and analyzed using TRFLP to resolve the microbial communities and their diversity based on land use, seasonal and temporal shifts. Surface water samples were analyzed for a human-specific genetic marker for Bacteroides spp. using the 16s rRNA gene. The concentrations of total and fecal coliforms during dry flows were comparable to wet flows at many of the sites along Chino Creek. Dendritic analyses of the microbial populations using TRFLP patterns demonstrated distinct clusters correlated with land use or potential flow from a location upstream. Bacteroides spp. was detected at a few of the sites, including effluent from two different treatment plants, but was not detected in reference water samples from the open-space location.