Neuropogonoid species in the lichen-forming fungal genusUsneaexhibit great morphological variation that can be misleading for delimitation of species. We specifically focused on the species delimitation of two closely-related, predominantly Antarctic species differing in the reproductive mode and representing a so-called species pair: the asexualU.antarcticaand the sexualU.aurantiacoatra. Previous studies have revealed contradicting results. While multi-locus studies based on DNA sequence data provided evidence that these two taxa might be conspecific, microsatellite data suggested they represent distinct lineages. By using RADseq, we generated thousands of homologous markers to build a robust phylogeny of the two species. Furthermore, we successfully implemented these data in fine-scale population genomic analyses such as DAPC and fineRADstructure. BothUsneaspecies are readily delimited in phylogenetic inferences and, therefore, the hypothesis that both species are conspecific was rejected. Population genomic analyses also strongly confirmed separated genomes and, additionally, showed different levels of co-ancestry and substructure within each species. Lower co-ancestry in the asexualU.antarcticathan in the sexualU.aurantiacoatramay be derived from a wider distributional range of the former species. Our results demonstrate the utility of this RADseq method in tracing population dynamics of lichens in future analyses.