Abstract: The complete sequences of Cyt b genes in Acanthogobius hasta and Odontamblyopus rubicundus were amplified, cloned and sequenced. The Cyt b genes in both species are1 141 bp in length. Comparing the obtained sequences, we detected 266 nucleotide variation sites, finding more transitions than transversions. Most base substitutions occur at the 3rd locus of the codon. Based on the complete Cyt b sequences of Gobiidae fishes recorded in GenBank，the genetic distance among 10 Gobiidae species calculated by Kimura-2 parameters model ranges from 0.118 to 0.330. The distance between Neogobius kessleri and N.gymnotrachelus is the shortest, being 0.118, while the distance between N.kessleri and A.hasta is the farthest, being 0.330. The phylogenetic analysis based on Cyt b gene sequence using NJ method suggests that the 10 Gobiidae fishes are divided into 3 main groups. Specifically, O.rubicundus and A.hasta cluster into one clade, having a relative far genetic distance to the other 8 Gobiidae species.