Interspecific hybridization and introgression can lead to partial genetic homogenization at certain neutral loci between morphologically distinct species and may obscure the species delimitation using nuclear genes. In this study, we investigated this phenomenon through population genetic survey of two alpine plants (Gentiana siphonantha and G. straminea) in the Qinghai-Tibet Plateau, where the distributions of two species are partly overlapped. We identified two clusters of chloroplast DNA haplotypes which correspond to the two species, and three clusters of ITS ribotypes. In addition to clusters specific to each species, the third ITS cluster, which was most likely derived from hybridization between the other two clusters and subsequent recombination and concerted evolution, was widely shared by two species in their adjacent areas. In contrast to the morphological distinctiveness of the two species, interspecific gene flow possibly led to genetic homogenization at their ITS loci. The new ITS lineage recovered for species in adjacent areas is distinctly different from original lineages found in allopatric areas. These findings may have general implications for our understanding of cryptic changes at some genetic loci caused by interspecific gene flow in the history, and they indicate that species delimitation should be based on a combination of both nuclear and chloroplast DNA sequence variations.