Abstract: To investigate genetic diversity of Mastacembelus armatus in Southern China, inter-simple sequence repeat (ISSR) was used to analyze eleven populations of 226 individuals from Fujian, Guangdong, Guangxi, Yunnan and Hainan provinces. 10 ISSR primers were used to amplify DNA and 112 loci were detected. 95 of 112 loci were polymorphic, and the percentage of polymorphic loci (PPL) was 84.82% at species level. The genetic diversity decreased in the order of EP HH TC YD HC LC ZC LY WH RH BS. The total Nei's genetic diversity (h) was 0.2126, and the Shannon's information index (I) was 0.3358, indicating a relatively high diversity of M. armatus. The total coefficient of genetic differentiation (GST) was 0.4620, meaning a 46.2% genetic variation indwelled among populations. The value of Nm was 0.5823, indicating lack of effective gene exchange among the populations, and genetic drift was the main factor of genetic differentiation among populations. The phylogeny tree showed that populations of the Dongjiang River and the Hanjiang River were clustered in one group and populations of the Xijiang River and the Beijiang River were clustered in another group, and populations of mainland were clustered in a big group and then joined to TC population.