The big-headed turtle (Platysternon megacephalum) is critically endangered because of overharvesting, illegal trade, and habitat destruction. Assessment of genetic variability in existing populations becomes very important to the taxonomy and conservation of this species. Here we describe 14 microsatellite loci isolated from an enriched genomic library of the big-headed turtle, and the polymorphisms of these loci were assessed in 28 individuals from Huizhou, Heyuan, Zhaoqing, and Shaoguan of Guangdong, China. The range of polymorphism information content is 0.305-0.738, and no evidence of significant linkage disequilibrium was found among any pairs of loci. These 14 new polymorphic microsatellite loci can be used in population genetics, taxonomy, phylogeography, behavior ecology, and conservation efforts of Platysternon megacephalum.