The mudskipper(Boleophthalmus pectinirostris) is an amphibious fish, adapting to a wide range of temperature and salinity. It inhabits the mudflats of intertidal zone and the mangrove forests in China, Korea, Japan, Vietnam and Malaysia. In China, it is mainly found in coastal mudflats of Zhejiang, Fujian, Taiwan, Guangdong and Guangxi provinces. Due to its economic importance, this species is widely cultured in southeast China. In the past 30 years, its natural resource has decreased gradually due to overfishing, loss and fragmentation of its natural habitats and environmental pollution. Genetic diversity of this species has been previously studied, but the samples were only obtained from a certain range of its habitats. In this study, amplified fragment length polymorphism(AFLP) markers were used to analyze the genetic diversity of 8 B. pectinirostris populations from the coastal waters of Northwest Pacific, in an attempt to illustrate its phylogeographic patterns and genetic structure. The findings of this study will provide insights into the future fishery management and conservation for B. pectinirostris. Instead of the traditional method polyacrylamide gel electrophoresis (PAGE), QIAxcel DNA High Resolution Kit Gel Cartridges was used to perform the AFLP analysis. Among the 126 individuals from the 8 populations in Japan, Korea, Vietnam and Guangxi, Guangdong, Fujian, Taiwan, and Zhejiang provinces of China, 186 loci between 15 bp and 500 bp were detected using 6 selective amplification primers(E-AGC/M-CTG, E-AGC/M-CCA, E-ATG/M-CCA, E-ATG/M-CTT, E-ACT/M-CAC, E-AGG/M-CTT), and 160 of which were polymorphic (86.02%). The amplified bands ranged from 25 to 36 for each primer combination. On the other hand, the detecting rates of each primer varied from 52.0% to 96.42%, with E-ACT/M-CAC showing the lowest detecting rate of 52.0% while other primers were all above 82%. The proportion of polymorphic loci from 8 populations varied from 27.96% to 57.53%, with the highest one for Korea population and the lowest one for Guangdong(China) population. The polymorphic loci proportion of B. pectinirostris were much lower than those of other fish species. Overfishing, habitats loss and environmental pollution may be the reasons for the low genetic diversity of B. pectinirostris. Nei’s distances between populations were from 0.038 to 0.151. The highest genetic distance was observed between Taiwan and Guangxi populations while the lowest was observed between Zhejiang and Guangdong populations. The pairwise comparisons of Fst ranged from 0.175 to 0.459, all of which were statistically significant(P=0). NJ tree constructed on the base of genetic distances between populations showed that the 8 populations were divided into two groups (A and B). Group A included Japan, Korea and Taiwan populations while Group B included the populations from Zhejiang, Fujian, Guangdong, Guangxi Provinces and Vietnam. The analysis of molecular variance showed that 15.48% of the total genetic variance was contributed by the among groups variation (Fct=0.15, P<0.01), 62.06% within populations variation (Fst=0.38, P=0) and 22.46% among populations within groups variation (Fsc=0.27, P=0). Coefficient of population differentiations (Fst) was 0.385 7 and the geneflow (Nm) from Gst was 0.796 4, which were comparatively lower than those of most other fish species. Also, strong genetic divergence between the populations of B. pectinirostris was found based on the pairwise comparisons of Fst and Nm. In comparison with the other marine migration fish, B. pectinirostris live in burrows on the mudflats of intertidal zone and do not have the feeding and spawning migration habits in their life cycle. Accordingly, their dispersal ability is very poor, leading to genetic differentiation between their populations. Since Japan, Korea and Taiwan are islands or peninsula, the habitats of B. pectinirostris populations in these locations are geographically different from those of the continental populations. Hence, genetic exchanges among them are very difficult to realize.