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Background: Geographic isolation caused by high-altitude valleys promotes the formation of geographic segregation of species, leading to species differentiation. The subgenus Oreosaura contains viviparous species from the Tibetan Plateau and the vicinity of the Tarim Basin, which can be divided into three species complexes according to their geographical distribution: Phrynocephalus vlangalii, Phrynocephalus theobaldi, and Phrynocephalus forsythii. However, molecular data for the P. forsythii complex are limited and the diversity of this species complex has been greatly underestimated. Therefore, this study aimed to explore the species diversity of Oreosaura and species differentiation within the P. forsythii complex. Methods: We analysed the species diversity of Oreosaura by combining previous data, constructed a phylogenetic tree of the subgenus based on cytochrome c oxidase subunit I and 16S sequences, and estimated the divergence time. Results: The results suggest significant genetic differences between the Tarim Basin populations and adjacent mountain valley populations of the P. forsythii complex and that the combination of deep valley landscapes in the high mountains and ice-age events have contributed to the differentiation of the viviparous toad-headed agama lizard, which is a key factor in the phylogenetics of the P. forsythii complex. Furthermore, we identified a population collected from Wuqia County, Xinjiang, as a new species, Phrynocephalus kangsuensis sp. nov. The results will provide data for phylogenetic studies following the P. forsythii complex and help demonstrate that valleys promote the formation of Phrynocephalus species. [ABSTRACT FROM AUTHOR]