A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.