Abstract: In order to analysis the genetic structure and demographic history of Boleophthalmus pectinirostris, the random amplified polymorphic DNA (RAPD) technology and mitochondrial cytochrome b (Cyt b) gene sequences were used in the study. The results showed that there was high level genetic diversity in the populations of B. pecinirostris in Yangtze River estuary and its southern wet-land adjacent regions. (1) RAPD was conducted to analysis the genetic variation of B. pecinirostris in Jiuduansha (JDS), Dinghai（DH）and Xiapu（XP）for 20 individuals per population, with 30 polymorphic 10-based random primers selected from 100 ones. In total, 236, 270 and 274 bands were obtained for JDS, DH and XP, respectively and there was 301useful loci. The range of proportion of polymorphic loci (P), Nei’s gene diversity (H) and Shannon diversity index (I ) was: 93.02%–96.3%, 0.3890–0.4219, 0.5618–0.6044, respectively. The results of AMOVA showed significantly genetic differentiation (F_st=0.03004-0.03254, P<0.05) but there was only 3.2% genetic variation among populations. (2) Results of Cyt b gene: the total Cyt b gene length was 1 141 bp and 33 haplotypes were obtained from 42 individuals collecting in 4 different regions, which included JDS, DH, XP and Cixi (CX). The average haplotype diversity (h) and nucleotide diversity (π) was 0.9184, 0.0048, respectively. The high level of N_m values (14.50–30.79) and low F_st values (0.0043–0.07814), the average K2-P distances (0.0040–0.0056), as well as the AMOVA analysis (total F_st=0.0193, P>0.05) all revealed that the B. pecinirostris had no obviously geographical differentiation. However, the phylogenetic tree estimated by neighbor-joining (NJ) method indicated that the individuals of B. pecinirostris formed two monophyletic lineages, and the two lineages were not correlated with geographical populations. Neutrality tests, mismatch distribution and network analysis suggested a late Pleistocene population expansion for B. pecinirostris (0.057–0.023 million years ago).