The molecular genetic tools using non-invasive samples such as faeces have an important role for the monitoring of wildlife and identification of elusive and rare species of semi-aquatic vertebrates. However, under aquatic environmental conditions the faeces undergo physical changes that harm the direct identification by visual inspection and leads to the confusion of the deposits from different cohabitating species. Consequently, the need of a refinement of molecular procedures is suggested. In this study this task was focused on the study of the reliability of the PCR-RFLP method to easily distinguish the faeces of the desman (Galemys pyrenaicus) from ecologically related species, including N. a. anomalus, but using an in silico-RFLP strategy. In this work, the faecal samples were collected from 2013 to 2014 from the LIC “Sierra de Gredos y Valle del Jerte” in the Mountain Central System of Spain, where the southernmost European population of the Pyrenean desman still survives. One primer pair was designed to amplify a very short portion of the mitochondrial cytochrome b gene containing targets for the restriction enzymes Hinf I, Mbo I and Hpy CH4 V, because the combination provides species-specific identifications. Pyrenean desman or Mediterranean water shrews were undoubtedly identified in the collected faeces. Moreover, the degree of homoplasy was studied under different scenarios of the three enzymes combination. Finally, the in silico-RFLP profiles given by the three enzymes combination have contributed with valuable information for a successful discrimination of faeces of the Pyrenean desman from those derived of Neomys fodiens, Neomys anomalus, Nectogale elegans and Cinclus cinclus.