To examine algal virus (Phycodnaviridae) genetic diversity in freshwater environments, gene fragments were cloned and sequenced from a river and a reservoir in Colorado, USA, and 2 different lakes in Ontario, Canada using PCR methods that target a diverse subset of known Phycodnaviridae DNA polymerase genes. Numerous phycodnavirus gene sequences were obtained from every sample, and rarefaction analysis of the sequence libraries demonstrated that virus richness was variable among different sample locations, and among samples collected from the same location at different times. Phylogenetic analysis of the unique sequences from each sample indicated that most sequences from the same geographic region (i.e. Colorado or Ontario) clustered together, but several exceptions were also observed. Phylogenetic analysis also demonstrated that the sequences obtained were more closely related to sequences from cultivated marine phycodnaviruses belonging to the genus Prasinovirus than to those from cultivated freshwater phycodnaviruses from the genus Chlorovirus. Overall, phycodnavirus sequences originating from cultivated marine viruses and marine clone libraries were not genetically distinct from the freshwater phycodnavirus sequences reported in this study.