Aims: Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. Methods and Results: Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (< 130 cells 100 ml-1) in 57-79% of samples taken from five locations. By comparison, a culture-based method detected Campylobacter in 0-23% of samples. Water quality parameters such as total Escherichia coli were not highly correlated with Campylobacter levels, although higher pathogen concentrations were observed at colder water temperatures (< 10 degrees C). Strains isolated from river water were primarily nalidixic acid-susceptible Campylobacter lari, and selected isolates were identified as Campylobacter lari ssp. concheus. Campylobacter from wild birds (seagulls, ducks and geese) were detected at a similar rate using PCR (32%) and culture-based (29%) methods, and although Campylobacter jejuni was isolated most frequently, C. lari ssp. concheus was also detected. Conclusions: Campylobacter were frequently detected at low concentrations in the watershed. Higher prevalence rates using quantitative PCR was likely because of the formation of viable but nonculturable cells and low recovery of the culture method. In addition to animal and human waste, waterfowl can be an important contributor of Campylobacter in the environment. Significance and Impact of the Study: Results of this study show that Campylobacter in surface water can be an important vector for human disease transmission and that method selection is important in determining pathogen occurrence in a water environment.