Abstract: 【Objective】To analyze the phylogenetic relationship of South China tiger, Panthera tigris amoyensis , Siberian tiger, P.tigris altaica and Bengal tiger, P.tigris tigris . 【Method】The fragment sequences of three mitochondrial genes were achieved by sequencing the PCR clones. These included CO Ⅰ 750 bp, CO Ⅲ 737 bp and ND4561 bp based on the three gene sequences analyses with the DNAStar software and the DNAssit software. Their base substitutions, Kimura 2-parameter distances and the percentage differences among three tiger subspecies were obtained. At the same time, the three genes sequences had fewer mutation sites. In addition, the Kimura 2-parameter distances of tiger subspecies was studied, selecting the mtDNAs of cat, Felis catus, leopard, Acinonyx jubatus and clouded leopard, Neofelis nebulosa as outgroup. The Mega 4.0 was used to reconstruct the evolutionary relationship of South China tiger, Siberian tiger, Bengal tiger with a neighbor-joining method. 【Result and conclusion】 The results of the Kimura 2-parameter distances of tiger subspecies indicates that Siberian tiger is closer to Bengal tiger than to South China tiger. The results of the CO Ⅰ, CO Ⅲ and ND4 genes show that Siberian tigers and Bengal tigers evolve from South China tigers.