Abstract: Belonging to Cobitidae of Cypriniformes,the subfamily Botiinae is a group of small or middle freshwater fishes dwellingin slow to moderately swift waters, and many of them are common aquarium species1 The classification of Botiinae fishes is con2troversial among different authors1Fang(1936)and Nalbant(1963) divided Botiinae into two genera, Botia and Leptobotia, whileChen(1980) separated it into three, Botia, Parabotia and Leptobotia1 Up to now, there was no study conducted on the structureof mitochondrial DNA control region and the molecular phylogenetic relationship of the subfamily Botiinae1 In the present study,the structure of the control region sequences was analyzed for fourteen species representing the three genera of Botiinae1 Similarto other fishes, the control region of Botiinae is composed of three domains, the extended termination associated sequence(ETAS) domain, the Central conserved domain(CD) and the Conserved sequence block(CSB) 1 The ETAS domain is very rich(73.3 %) in the contents of A and T, while the CD domain is much rich(36. 3 %) in G and C1 A termination2associated se2quence TAS(TACATA)and its reverse complementary sequence(ATGTAT)were found in the ETAS domain, and the conservedsequence in this domain is ETAS 11 The CD domain is highly conserved, and three conserved sequences were identified, CSB2F, CSB2E and CSB2D1 Three conserved sequences were identified in the CSB domain, CSB21, CSB22 and CSB23.With Myxocyprinus asiaticus as outgroup, compared with members of Nemacheilinae, Cobitinae and Homalopteridae aswell, the phylogenetic relationship of Botiinae was analyzed using the neighbor2joining(NJ) method in MEG A2.1, the maximumparsimony(MP) method in PAUP34.0b10 and the maximum likelihood(ML) method in TREE2PUZZ LE 5.01 The results showedthat the subfamily Botiinae is monophyletic and the confidence levels in the three analysis methods were 88 %in NJ, 57 %in MPand 97 % in ML respectively. It also supported that Botiinae contains three genera, Botia, Parabotia and Leptobotia, sinceeach of these genera formed a monophyly, which were supported with 100 %, 65 %and 78 %of bootstrap values in NJ method and100 %, 75 %and 70 %of Quartet puzzling values in ML method1 In MP analysis, the bootstrap value supporting monophylyof thegenus Parabotia was less than 50 %, while for Botia and Leptobotia, the values were 100 %and 56 %.Based on the results of molecular phylogenetic analysis, morphological comparison and geographical distribution of Botiinaefishes, it was suggested that Botia is the most primitive genus, Parabotia the second, and Leptobotia the most specialized.