The population genetic variations and phylogeographical patterns of 107 Micronoemacheilus pulcher from 12 drainage systems in western South China were investigated based on 934-938 base pairs (bp) nucleotide sequences of mtDNA control region. We found 79 nucleotide sites variable in the full sequence. Analysis of molecular variance (AMOVA) revealed that genetic variation within the populations was 42.84%, among the populations was 58.53%. Thirty-six haplotypes of the mtDNA control region were analyzed using the neighbor-joining method. The populations of 12 drainage systems could be divided into two lineages. The drainages in Guangxi (Fangcheng River, Dongzhong River, Beilun River and Nanliujiang River) and Xijiang River were more closely related to Moyangjiang River and Tanjiang River, the Wanquanhe River and Nandujiang River in Hainan island were closely related to Jianjiang River in Guangdong Province. Nested clade phylogeographic analysis indicates that this fish has originated from the joining area of Guangxi and Vietnam (in which Dongzhong River, Beilun River and Fangcheng River are located) and spread to other drainages by two ways: (1) through Xijiang River spread to drainages of Guangxi, Moyangjiang River and Tanjiang River of Guangdong Province; (2) through ancient river systems between Hainan Island and Vietnam to Hainan Island, then back to the mainland expanding northward to Jianjiang River of Leizhou Peninsula in Guangdong Province. The current phylogeographical distribution of the populations might have formed by several events: allopatric fragmentation, long-distance colonization possibly coupled with subsequent allopatric fragmentation, contiguous range expansion.