Water samples were analysed to differentiate human and animal faecal contamination of the New River, Mexico/USA, by genotyping bacterial viruses detected in the samples. From 46 water samples collected from the New River, 372 plaques of male-specific coliphages were isolated and genotyped; 44% of the plaques were identified as F-RNA coliphages and further characterized into four groups. Group I was the most prevalent (56%), followed by group IV (25%), group III (10%) and group II (9%). Group III coliphages were only detected at the sampling site in the vicinity of the international boundary, indicating human faecal contamination. As the New River traverses through the US region, groups I and IV coliphages were predominantly identified, but no human-specific genotypes were detected. The study also found that water temperature influenced the prevalence of the relative proportions of F-RNA coliphages in the environmental water samples. The strategy used in this study appears to be a practical and reliable tool for monitoring and distinguishing between human and animal faecal contamination.