Hainan Island harbours an extraordinary diversity of Gesneriaceae with 14 genera and 23 species, among which two species and one variety are recognized in the genus Oreocharis. These three Oreocharis taxa are all Hainan-endemics and show complex geographical distribution pattern with considerable morphological intermixtures. In this study, we combined DNA (nuclear ITS sequences and cpDNA trnL-trnF and ycf1b) to evaluate genetic delimitation for 12 Oreocharis populations from the island, together with morphological similarity analysis using 16 morphological traits. The results showed Hainan Oreocharis taxa was monophyly with relative low genetic diversity within populations, highly significant genetic differentiation among populations and a significant phylogeographical structure. The 12 populations formed three genetically distinct groups, roughly correspondent to the currently recognized two species and one unknown lineage. The PCA analyses of morphological traits indicate three distinctive groups, differing mainly in petal color and corolla shape and types. The roles of river isolation in the origin and distribution of these three lineages are discussed.