Plateau pika (Ochotonidae: Ochotona curzoniae) is widespread in Qinghai-Tibetan Plateau (QTP). It is endemic to QTP and restricted to high altitude area, ranging from 3,000 to 5,200 m above sea level. In this study, I examined the genetic structure of O. curzoniae along the Qinghai-Tibetan Railway (QTR) using mitochondrial cytochrome b sequences, and investigated whether QTR had become a barrier of gene flow to populations of O. curzoniae on its two sides. In total, I found 26 haplotypes of cyt b sequences from 29 individuals, of which the mean length was 1,190 bp including 68 informative sites, and the mean genetic distance was 1.7%. Two major clades were revealed in phylogenetic trees as well as TCS haplotype network. In general, the relative positions of haplotypes in the clades were corresponded to their geographic distribution. Most haplotypes of clade I were from east side, while all the haplotypes of clade II were from west side of QTR. Significant population differentiation was revealed between populations from the opposite sides of QTR, but not the same side. Due to the far smaller geographical distances between sampling sites between opposite sides than those of the same side, the differentiation pattern was not consistent with the theory of isolation by distance. Therefore, QTR might have resulted as a barrier to gene flow between populations of O. curzoniae living on opposite sides of QTR. The degree of genetic differentiation between populations of O. curzoniae on opposite sides of QTR will probably further increase in the future.