Bighead carp （Hypophthalmichthys nobilis） is a common freshwater fish species in China. In order to reveal the genetic status of H. nobilis in the middle and lower reaches of the Yangtze River （YR）, 219 individuals were collected from five locations respectively in Shishou （SS）, Changsha （CS）, Ruichang （RC）, Yangzhou （YZ） and Zhangjiagang （ZJG）. The D-loop sequences were tested and analyzed in comparison with those of H. nobilis from the Pearl River （PR, 213 individuals） and Northern America （NA, 33 individuals） downloaded from the NCBI. As a result, 35, 96, and 11 variable sites were observed and 37, 62, and 3 haplotypes were defined in YR, PR, and NA, respectively. Among the YR H. nobilis, high haplotype diversity （H_d, 0.798-0.897） and moderate nucleotide diversity （π, 0.003 38-0.006 53） were detected, except the pairwise pairs of RC vs. ZJG （F_ST = 0.012, P>0.05） and CS vs. YZ （F_ST = 0.018, P>0.05）, significant genetic differentiations （F_ST ranged from 0.031 to 0.125, P<0.05） were found among all the other pairwise stock pairs, and gene flows （N_m ranged from 3.509 to 39.993） were generally detected within the five stocks. According to the haplotype network analysis, the D-loop haplotypes contained multiple clades which were obviously shared among different stocks in YR. Meanwhile, the central haplotypes of YR and PR populations were generally mutual-shared, whereas the haplotypes specific to PR were peripherally distributed. The haplotypes sharing situation further evidenced the possible multiple introductions of NA. Based on the neutrality test, it was predicted that only PR went through population expansion in history. Overall, the study revealed an abundance of genetic variations in H. nobilis of YR and PR, laying a solid foundation for the further protection and utilization of the relevant genetic resources.