To date, the origins, domestication, and genetic structure of Chinese Mongolian horses (CMH) are poorly understood. Furthermore, there have been sparse reports on the genetic differences between CMH and Thoroughbred. In order to determine their genetic structure, understand their genetic relationships, and explore their domestication processes, we performed an extensive survey of creatine kinase (muscle isoenzyme; CKM) variations among six populations of indigenous CMH, cultivated Sanhe horses, and imported Thoroughbred. Twenty-three single-nucleotide polymorphisms were found among the 343 horse sequences. From these, 40 haplotypes were inferred. Haplotype diversity (H) values differed from 0.6424 to 0.7881 and nucleotide diversity (π) values ranged from 0.00150 to 0.00211. The differences between Thoroughbred population and other Chinese horse populations were large, but only small differences were observed among Chinese horse populations with respect to CKM intron sequences suggesting that the domestication history, breeding measures, and origins of these horse populations are completely different. Results suggest that Sanhe and CMH are very closely related and the introgression (interbreeding) between them is serious. Our results suggest that Sanhe and Wushen require prompt and powerful protection. Overall, CKM intron was an appropriate marker for the determination of genetic relationships among horse populations and breeds.