Farmed animals such as sheep, cattle, swine and poultry play an important role in microbial contamination of water, crops and food, and introduce large quantities of pathogens into the environment. The ability to determine the origin of faecal pollution in water resources is essential when establishing a robust and efficient water management system. Animal-specific viruses have previously been suggested as microbial source tracking tools, but specific ovine viral markers have not been reported before now. Previous studies have shown that polyomaviruses are host-specific, highly prevalent and are commonly excreted in urine. Furthermore, they have been reported to infect several vertebrate species but not sheep. That situation encouraged the study of a new putative ovine polyomavirus (OPyV) and its use to determine whether faecal pollution originates from ovine faecal/urine contamination. Putative OPyV DNA was amplified from ovine urine and faecal samples using a broad-spectrum nested PCR (nPCR). Specific nested PCR and quantitative PCR assays were developed and applied to faecal and environmental samples, including sheep slurries, slaughterhouse wastewater effluents, urban sewage and river water samples. Successful amplification by PCR was achieved in sheep urine samples, sheep slaughterhouse wastewater and downstream sewage effluents. The assay was specific and was negative in samples of human, bovine, goat, swine and chicken origin. Ovine faecal pollution was detected in river water samples by applying the designed methods. These results provide a quantitative tool for the analysis of OPyV as a suitable viral indicator of sheep faecal contamination that may be present in the environment.