Abstract: In order to explore the genetic structure and differentiation degree among different Ptychidio jordani populations in Xijiang River flowing through Guangxi Province, we collected 139 individuals from six wild P. jordani populations (HSH, LIUJ, XIJ, YOUJ, YUJ, ZJ) and analyzed their genetic structure with Cytb sequences. The length of Cytb gene was 1 053 bp, and the average contents of T, C, A and G were 29.1%, 27.7%, 29.3% and 13.9%, respectively. Besides, the A+T content (58.4%) was significantly higher than the C+G content (41.6%). A total of 20 haplotypes were defined, clustering into two branches without significant geographic clusters. The average haplotype diversity and nucleotide diversity of the six populations were 0.768 2 and 0.002 3, respectively. The highest genetic diversity was detected in HSH population (h=0.748 7, π=0.003 3), while the relatively low genetic diversity was found in LIUJ (h=0.274 4, π=0.000 4) and ZJ (h=0.374 7, π=0.000 3). The genetic differentiation index (F_ST) of six P. jordani populations was 0.461 4 (P<0.001), showing a great genetic differentiation. The degree of genetic differentiation between ZJ and LIUJ populations was the greatest, while that between LIUJ and XJ populations was the smallest. AMOVA analysis shows that part of the genetic variation was within the populations (53.86%), and part came from different populations (46.14%). Neutral test (Tajima's D=−1.082 8, P>0.05; Fu's Fs=−6.572 5, 0.01<P<0.05) and base mismatch distribution analysis show that the P. jordani population in Xijiang River experienced population expansion 0.07−0.187 million years ago (Ma). In conclusion, the genetic diversity of P. jordani between LIUJ and ZJ populations is low in Guangxi Xijiang River; the total population differentiation degree is high, but still belongs to a population, and the spatial distance and geographical barrier can promote the genetic differentiation.