In this study,mitochondrial cytochrome b(Cyt b) gene sequencing and random amplified polymorphic DNA(RAPD) technique were applied to study the phylogenetic relationships of four species of genus Macropodus: M.opercularis,M.chinensis,M.hongkongensis and M.erythropterus.The complete sequences of the mitochondrial Cyt b gene(1 155 bp) of 4 species from 14 individuals were sequenced.Combined these sequences with homologous sequences from GenBank Database,M.opercularis and M.concolor were used as ingroup;Pseudosphromenus cupanus,P.dayi,Malpulutta kretseri and Batta splendens were used as outgroup.Moreover,36 random primers were selected from 133 primers,and 749 reproducible bands were amplified in 96 individuals from 10 populations under optimized reaction conditions.Molecular phylogenetic trees based on Cyt b gene sequences were constructed by neighbor-joining(NJ) and minimum evolution(ME) methods,respectively.The results indicated that M.hongkongensis was grouped with M.erythropterus firstly,then clustered with M.opercularis,and M.chinensis was in the outer edge.The UPGMA cluster analysis based on RAPD data was identical to the molecular phylogenetic trees based on Cyt b.The results reflect that: M.chinensis has a distant relationship with other fishes of genus Macropodus;genetic distances between species' were 0.184 5-0.225 3(Cyt b) and 0.653 6-0.746 5(RAPD);M.chinensis and other Macropodus represent two independently evolving natural groups of the same monophyletic group.Genetic differentiation among M.hongkongensis and M.opercularis was obvious,Cyt b sequence divergence between species' was 11.00%,genetic distance between species' was 0.577 7(RAPD),which agrees with the view that M.hongkongensis is a valid species.The genetic diversity between different M.hongkongensis populations was significant,Cyt b sequence divergence was 3.12%,genetic distance was 0.060 1(RAPD).Nei's gene diversity(H_E) and Shannon's Information index(I) between M.opercularis populations were 0.058 2 and 0.086 9,and among populations were 0.016 1-0.031 7 and 0.023 5-0.046 7,respectively,which show that the genetic diversity of M.opercularis mainly between populations too,and the individuals cluster respectively according to their distribution of river basin.