The hillstream catfish, Glyptothorax laosensis, is endemic to basin of Lancang River (also known as Mekong River). Genetic variation was detected within and among 8 populations of G. laosensis in Lancang River based on mitochondrial cytochrome b sequences. A total 16 nucleotides sites were detected in 1 138 bp fragment, which defined 15 haplotypes from 129 sequences. The variation level was very low, with mean h=0.299 and π=0.00032, respectively, and no variation was detected in some populations. Hierarchical analysis of molecular variance (AMOVA) showed no significant genetic structuring among populations. The unimodel mismatch distribution of pairwise haplotype difference and the negatively selective neutrality test (Tajima's D=-2.3696, P<0.02; Fu's Fs=-20.975, P<0.05) suggested that a recent population expansion had occurred.