Bupleurum commelynoideum H. Boissieu is a perennial herb occurring throughout the Hengduan Mountains region. This species was found to have more than one chromosome number in our previous chromosomal study. For a better understanding of its intraspecific genetic differentiation and interspecific relationships with other Bupleurum members, phylogenetic relationships among 15 populations of B. commelynoideum and 22 other Bupleurum members were investigated using the nuclear ribosomal DNA internal transcribed spacer (ITS) region and three chloroplast DNA (cpDNA) fragments (trnL-F, rps16, and rpl32-trnL). Both phylogenetic analyses of the ITS region and combined cpDNA data indicated that B. commelynoideum has more than one geographically delimited lineage. Among them, accessions from Shangri-La and other regions were considered as different cryptic species according to the unified species concept. Our analyses also revealed that a complex pattern of reticulate relationship exists in B. commelynoideum and its allied species. The chromosome numbers of four populations of B. commelynoideum and a closely related species B. rockii H. Wolff are reported. The results showed that the chromosome number of B. commelynoideum has abundant variations in both chromosome ploidy (2x; 4x; 6x) and basic chromosome number (x = 5; x = 6). Population DXS (x = 6), which has the different basic chromosome number, should represent a different kind of cryptic speciation in B. commelynoideum. The karyological evidence, together with the molecular data, indicated that the existence of at least three cryptic species in B. commelynoideum. This kind of genetic differentiation without morphological divergence might be caused by stabilizing selection imposed by the extreme environmental conditions of the alpine area.