The genome compositions and genetic relationships of seven species of Kengyilia were assessed using a sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) technique. Five species, K. kokonorica, K. rigidula, K. hirsuta, K. grandiglumis, and K. thoroldiana, are native to Qinghai (China). The other two, K. alatavica and K. batalinii, are distributed in Xinjiang (China) and Kyrgyzstan, respectively. Each chromosome could be easily identified using chromosome markers (45S rDNA, 5S rDNA, pAs1, and AAG repeats) by FISH and allocated to the St, P, or Y genome by GISH. Molecular karyotype comparison indicated that K. alatavica and K. batalinii were distinct from the Qinghai species in all three genomes. These results support that the species of Kengyilia from Central Asia and the Qinghai-Tibetan plateau have independent origins. Genomic differentiation was still detected among the species of Kengyilia from Qinghai. Specifically, a common species-specific pericentric inversion was identified in both K. grandiglumis and K. thoroldiana, and an identical St-P non-Robertsonian translocation was frequently detected in K. hirsuta. The Qinghai species formed three genetic groups, K. kokonorica-K. rigidula, K. hirsuta, and K. grandiglumis-K. thoroldiana. The possible role of species-specific inversions and translocations in the evolution of StPY species is discussed.