The wild water buffalo is highly endangered, with the few remaining populations already affected or likely to be increasingly affected by hybridization with domestic buffalo. The work described here was done to evaluate a genetic method to discriminate wild from mixed ancestry (hybrid) and domestic animals, and to identify with high probability those most likely to be purebred wild. Samples from 45 animals (phenotypically classified into three groups - ten wild, 28 domestic and seven hybrid) were genotyped for ten microsatellite loci. Although genetic distances among the three groups were small, an assignment test identified two of the ‘wild’ and seven of the ‘domestic’ as hybrids. However, sample sizes also are small, indicating the need for a conservative approach in the first instance in using these results. As more animals are genotyped, assignments will become more accurate, and a translocation programme to establish a second Nepalese wild population in a protected area could be undertaken.