AimsMolecular subtyping is commonly used in foodborne disease surveillance and microbial source tracking. There is a knowledge gap regarding the molecular ecology of foodborne pathogens in non-food-associated environments. The objective of this study was to isolate and subtype foodborne pathogens from pristine natural environments with minimal anthropogenic inputs. Materials and ResultsFive locations (wilderness areas) in Northern Colorado were sampled during the spring, summer and fall over a 2-year period. Soil, water, sediment, surface soil and wildlife faecal samples were microbiologically analysed to detect Listeria, Salmonella and Shiga toxin-producing Escherichia coli (STEC), and resultant isolates were subtyped. Three samples tested positive for Listeria monocytogenes and 19 samples contained other Listeria spp. Salmonella was isolated from two samples, five samples contained non-O157 STEC, and E.coli O157:H7 was not detected. Two L. monocytogenes isolates from faecal samples collected from the same wilderness area over a year apart shared the same PFGE pattern, while all other isolates had a unique type. ConclusionsOur data indicate that (i) there was a rare presence of human foodborne pathogens in pristine natural environments in Northern Colorado, (ii) there was genetic diversity between organisms isolated within a given wilderness area, and (iii) the Northern Colorado climate and topography may contribute to the low occurrence of these organisms. Significance and Impact of the StudyRelatively little is known about the molecular ecology of foodborne pathogens in pristine natural environments. While foodborne pathogens were rarely detected in wildlife faecal and environmental samples from the wilderness areas in this study, some isolates shared DNA fingerprint types with human clinical isolates from same region during the same time frame, highlighting the need for environmental isolate subtype data. The availability of molecular subtyping data for non-food-associated foodborne pathogen isolates can facilitate epidemiological and microbial source tracking investigations.