Aims To investigate the prevalence, seasonality and genetic diversity of Salmonella enterica serotypes, particularly those of human and veterinary health significance, in urban and rural streams. Methods and Results Using a swab collection technique and multiple culture media for isolation, Salmonella were detected in 78 center dot 4% of water samples (November 2003 to July 2005) taken from urban and rural/agricultural streams in the Grand River watershed (Ontario, Canada). Among 235 isolates, there were 38 serotypes, with the predominant serotypes and phagetypes (PT) being Salmonella Typhimurium PT 104 and Salmonella Heidelberg PT 19. These are also the most common Salmonella serotypes found in humans and farm animals locally and across Canada, a trend not commonly reported. The urban stream had more frequent Salmonella occurrence, greater serotype diversity and greater genetic variability (based on pulsed field gel electrophoresis) of specific strains compared with the rural/agricultural streams. Distinct seasonality in serotypes of health significance was observed only in the rural/agricultural streams, which is likely a reflection of seasonal source inputs in these watersheds. Despite the lower occurrence of these strains in stream water in the colder months, laboratory studies did not support reduced survival of Salm.Typhimurium and Salm.Heidelberg at lower temperatures, although survival differences were observed with other serotypes. Conclusions A diverse range of Salmonella serotypes and PT were obtained from both urban and rural/agricultural streams, with the predominant strains being those most frequently associated with human and veterinary disease in Canada. Significance and Impact of the Study The ubiquitous nature of Salmonella in water and the predominance of serotypes/PT of human or veterinary health significance suggest that the aquatic environment is a reservoir for this bacterium and could be involved in the transport and dissemination of this pathogen between hosts.