In this study 13 Chondrostoma regium specimens from the Leileh, Armand, Marbor, Shalamzar, and Koohrang Rivers in Zagros region were compared to other species of Chondrostoma and some species of the Leuciscinae subfamily. A total of nine 652 bp haplotypes were recovered. Results showed that C. regium in Karoon basin is a monophyletic group which is different from its counterpart in the Leileh River in Kermanshah Province. In addition, the highest genetic distance among the observed haplotypes was for Leileh-Armand and Leileh-Marbor Rivers (0.8%).  The time elapsed since the isolation of C. nasus and C. kinzelbachi from C. regium and the isolation time between C. regium and C. lemmingii were calculated as about 2-3 MY and16 MY, respectively