Blue oat mite species, including Penthaleus major and P. tectus, are pests widely distributed across China that cause damage to winter wheat. This study evaluated the genetic diversity of P. major and P. tectus on Triticum hosts collected from 23 geographic locations based on mitochondrial cytochrome c oxidase subunit I (COI) sequences. We identified nine haplotypes in 438 P. major individuals from 21 geographic locations and five haplotypes in 139 P. tectus individuals from 11 geographic locations. Meanwhile, P. major exhibits high values of haplotype diversity (Hd) and nucleotide diversity (Pi) (Hd = 0.534 > 0.5 and Pi = 0.012 > 0.005), representing a large stable population with a long evolutionary history. P. tectus shows low values of Hd and Pi (Hd = 0.112 < 0.5 and Pi = 0 < 0.005), which suggest recent founder events. Moreover, demographic analysis suggested that P. major and P. tectus have not undergone a recent population expansion. The lowest genetic variation was observed in Xiangzhou (XZ-HB), Zaoyang (ZY-HB), Siyang (SY-JS), and Rongxian (RX-SC), with only one species and one haplotype identified in over 30 individuals. Robust genetic differentiation was found in P. major compared to P. tectus, which provides a theoretical basis for the widespread distribution of P. major in China.