The giant panda Ailuropoda melanoleuca is a critically endangered species endemic to China. In order to carry out effective genetic management for the giant panda population, sufficient and reliable polymorphic genetic markers are required to provide essential information on the genetic diversity survey of this species. Seven new tetranucleotide microsatellite loci were isolated and characterized in this study and presented here as a tool for evaluating the genetic variation of giant pandas in the world's two largest captive populations (Chengdu Research Base of Giant Panda Breeding, Sichuan Province and the China Research and Conservation Center for the Giant Panda in Wolong, Sichuan Province). A total of 45 alleles were identified from these seven new microsatellite loci on the basis of 48 giant panda individuals, including 19 private alleles (six from the Chengdu population, 13 from the Wolong population) and 26 shared alleles. The average number of alleles, the average allelic richness the and mean observed heterozygosity were 4.6, 4.367 and 0.649, respectively in the Chengdu population and 5.6, 5.697 and 0.675 in the Wolong population, suggesting that the Chengdu population has a much lower allelic diversity than the Wolong population. Thus, we proposed a better strategy for the captive breeding of giant pandas.