There are only six isolated living giant panda populations, and a comprehensive understanding their genetic health status is crucial for the conservation of this vulnerable species. Liangshan Mountains is one of the main distribution areas of living giant pandas and is outside the newly established Giant panda national Park. In this study, 971 giant panda fecal samples were collected in the heartland of Liangshan Mountains (Mabian Dafengding Nature Reserve: MB; Meigu Dafengding Nature Reserve: MG; and Heizhugou Nature Reserve: HZG). Microsatellite makers and mitochondrial D-loop sequences were used to estimate population size and genetic diversity. We identified 92 individuals (MB: 27, MG: 22, HZG: 43) from the three reserves. Our results showed that: 1) Genetic diversity of three giant panda populations was medium-low; 2) Quite a few loci deviated significantly from the Hardy-Weinberg equilibrium and almost all these deviated loci showed significant heterozygote deficiencies and inbreeding; 3) Three giant panda populations have substantial genetic differentiation with the most differentiation between MB and the two other populations; 4) a large amount of giant panda feces outside the three reserves were found, implying the existence of protection gap. These results indicated that the giant panda population in Liangshan Mountains is at an risk of genetic decline or extinction given stochastic events and urgent need of human management. This study revealed that high attention should be paid to the protection of these giant panda populations outside the Giant panda national Park, to ensure them survival in their distribution areas.