Accurate species delimitation is crucial to the fields of systematics, biogeography, and conservation biology; however, inferring phylogenetic relationships among closely related, recently diverged species has always been difficult. In the present study, we conducted a range-wide field investigation of five Pedicularis species of P. sect. Cyathophora, which includes all four basic corolla types present in the genus. We reconstructed the phylogeny of this section by using three single/low-copy nuclear genes (CRC, LFY-L, LFY-S) and two chloroplast genes (matK, ycf1). To clarify the evolutionary history of sect. Cyathophora, we also estimated divergence times of the genus Pedicularis. Additionally, we evaluated the phylogenetic utility of nrDNA ITS in sect. Cyathophora by cloning and sequencing analyses of the ITS region for all of the investigated samples. The results of our multilocus phylogenetic analysis supported the delimitation of three species of sect. Cyathophora, but failed to support the definition of P. rex and P. thamnophila. On the basis of morphological, geographic, genetic, and phylogenetic evidence, we suggest to include P. thamnophila in P. rex. The molecular dating analysis showed that the uplift of the Qinghai-Tibet Plateau and climatic changes may have played important roles in the divergence and speciation of sect. Cyathophora. The results of clone-based sequencing revealed that extensive nrITS polymorphisms exist in the individuals of ingroups; these polymorphisms may cause misleading phylogenetic inference for sect. Cyathophora.