Frequent discordant phylogenies inferred from different loci, as well as the presence of sufficiently diverged gene variants within a single species isolate are indicative of potentially frequent non-monophyly in the genus Pinus. Interspecies hybridisation and incomplete lineage sorting have been suggested as possible explanations for the observed phylogenetic discrepancies. However, there is no direct evidence to support any of the proposed scenarios for the Eurasian five-needle pines. We used natural hybrids between Pinus sibirica and P. pumila, as well as their parental species, as a model to reproduce the scenario of non-monophyly in the subgenus Strobus. Three non-linked nuclear DNA loci (LEA, AGP6 and 4CL) were applied to detect introgressive alleles and to genetically discriminate the studied species. Comparative sequence analyses revealed two clusters of species-specific alleles for each of the markers, characteristic for either P. sibirica or P. pumila. No hybrid-specific alleles were found. We also found no hybrids with a genotype characteristic of only one of the parental species for all three loci. On average, the hybrids were characterised by an equal ratio of alleles from the P. sibirica and P. pumila clusters. We reveal that some trees of pure species originating from allopatric locations have non-specific loci that can be a result of genetic exchange between these species in the distant past or incomplete lineage sorting.