We investigated the prevalence of sapoviruses (SaVs) in the Tamagawa River in Japan from April 2003 to March 2004 and performed genetic analysis of the SaV genes identified in river water. A total of 60 river water samples were collected from five sites along the river, and 500 ml was concentrated using the cation-coated filter method. By use of a real-time reverse transcription (RT)-PCR assay, 12 (20%) of the 60 samples were positive for SaV. SaV sequences were obtained from 15 (25%) samples, and a total of 30 SaV strains were identified using six RT-PCR assays followed by cloning and sequence analysis. A newly developed nested RT-PCR assay utilizing a broadly reactive forward primer showed the highest detection efficiency and amplified more diverse SaV genomes in the samples. SaV sequences were frequently detected from November to March, whereas none were obtained in April, July, September, or October. No SaV sequences were detected in the upstream portion of the river, whereas the midstream portion showed high positive rates. Based on phylogenetic analysis, SaV strains identified in the river water samples were classified into nine genotypes, namely, GI/1, GI/2, GI/3, GI/5, GI/untyped, GII/1, GII/2, GII/3, and GV/1. To our knowledge, this is the first study describing seasonal and spatial distributions and genetic diversity of SaVs in river water. A combination of real-time RT-PCR assay and newly developed nested RT-PCR assay is useful for identifying and characterizing SaV strains in a water environment.