Understanding population history and genetic structure are key drivers of ecological research. Here, we studied two highly fragmented and isolated populations (Xiaoxiangling and Daxiangling) of giant pandas (Ailuropoda melanoleuca) at the extreme southwestern edge of their distribution. This area also contains the Dadu River, national road 108 and various human infrastructure and development, providing an ideal region in which we can identify the effects of different barriers on animal movements. We used partial mitochondrial control region (mtDNA) and nine microsatellite loci (nuclear DNA) data derived from 192 faecal and one blood sample collected from the wild. We found 136 genotypes corresponding to 53 unique multilocus genotypes and eight unique control region haplotypes (653 bp). Significant genetic boundaries correlated spatially with the Dadu River (K = 2). We estimate that a major divergence took place between these populations 26 000 years bp, at around the similar time the rock surface of valley bottom formed in Dadu River. The national road has resulted in further recent population differentiation (Pairwise FS on mtDNA and nuclear DNA) so that in effect, four smaller sub-populations now exist. Promisingly, we identified two possible first-generation migrants and their migration paths, and recommended the immediate construction of a number of corridors. Fortunately, the Chinese government has accepted our advice and is now planning corridor construction.