Abstract: The species range of Boleophthalmus pectinirostris sensu lato includes two disjunctive areas, i.e., East Asia and Strait of Malacca in Malaysia along the northwestern Pacific coast. However, the species status of Malaysian populations remains disputed. Mitochondrial ND5 gene (718 bp) and nuclear Rag1 gene (1395 bp) were used to reconstruct phylogenetic relationships among Boleophthalmus pectinirostris fishes by sampling 45 specimens from 11 locations in the northwestern Pacific. The results showed that Boleophthalmus pectinirostris fishes could be divided into two major monophyletic groups, i.e., East Asian lineage and Malaysian lineage, and which together formed the sister-group relationship. Species delineation using the analyses of GMYC and *Beast species tree supports that East Asian lineage and Malasian lineage of Boleophthalmus pectinirostris sensu lato should be placed into two different species. Molecular dating revealed that the divergence time between East Asian lineage and Malaysian lineage of Boleophthalmus pectinirostris sensu lato was 2.73 Ma. We concluded that Boleophthalmus pectinirostris sensu lato was a complex, including two species. The East Asian populations is Boleophthalmus pectinirostris sensu stricto, and the Malaysian populations is a cryptic species (Boleophthalmus sp.). Our findings suggested that species split between Boleophthalmus pectinirostris sensu stricto and Boleophthalmus sp. was attributed to geographical isolation during lowing sea levels of ice ages and the barrier of gene flow induced by ocean currents during interglacial period in the late Pliocene.