The mouflon (Ovis gmelini ophion) is the largest wild terrestrial mammal of Cyprus. Regarded as the local flagship species, its population range is limited to the mountainous Paphos Forest and adjacent areas including part of Troodos National Forest Park (> 700 Km2). This species, protected by both national and international legislation, is classified as Endangered by the IUCN, with livestock intrusion, poaching and habitat loss as the main threatening factors. We determined the spatial genetic structure by investigating sexes separately and shed further light on the Cyprus mouflon placement among Ovis haplogroups (HGs) to enforce its protection within an adaptive conservation framework. We genotyped 108 samples collected across the entire species’ range at both14 loci of the microsatellite DNA and the mitochondrial DNA Control Region (CR). Microsatellite genotyping indicated that the wild population retained low genetic variability, which, however, was not associated with a level of inbreeding raising particular concern (FIS = 0.12). An overall weak spatial genetic structure was disclosed, consistently with the limited extension of the mouflon range, the lack of significant physical barriers to dispersal and the intense gene flow mostly occurring along a northeast-southwest axis across the forest. Nevertheless, evidences of structure were found for females in compliance with their philopatric behaviour. The disclosure of unique features compared to Mediterranean and Near East conspecifics, such as the occurrence of a six-fold 76 bp-long repeated motif in the Cyprus mouflon CR, along with the outcome of a phylogenetic reconstruction (based on a far higher number of Ovis GenBank records and Cyprus haplotypes than in previous studies) inspired the proposal of a new haplogroup (HGC2) which included the Anatolian mouflon (O. g. anatolica, former HGX) as sister taxon. While both high habitat heterogeneity and low human disturbance were the main drivers in determining the overall spatial structure, future conservation efforts to preserve this valuable genetic resource should focus on avoiding possible introgressive hybridisation with co-grazing livestock to the edge of its range.