The guigna (Leopardus guigna) is the smallest and most-restricted New World cat species, inhabiting only around 160,000 km(2) of temperate rain forests in southern South America and is currently threatened by habitat loss, fragmentation and human persecution. We investigated phylogeographic patterns of genetic diversity, demographic history and barriers to gene flow with 116 individuals sampled across the species geographic range by analyzing 1,798 base pairs of the mtDNA (496 bp HVSI region, 720 bp NADH-5 gene, 364 bp from 16S gene and 218 bp from ATP-8 gene) and 15 microsatellite loci. Mitochondrial DNA data revealed a clear phylogeographic pattern with moderate separation between northern and southern Chilean populations supporting recognized subspecific partitions based on morphology. A recent demographic expansion was inferred for the southern-most group (San Rafael Lake), presumably due to the complete coverage of this area during the last glacial period, 28000-16000 years BP. Geographical barriers such as the Andes Mountains and the Chacao Channel have partially restricted historic and more-recent gene flow and the Chilo, Island population has diverged genetically since being separated from the mainland 7000 years BP. This is the first study of the genetic structure of this threatened species throughout its whole geographic range.