Chinese alligator (Alligator sinensis) is an endangered freshwater crocodilian endemic to China, which experienced a severe bottleneck about 30 years ago. In this study, we developed locus-specific primers to investigate the polymorphism of 3 major histocompatibility complex (MHC) loci in 3 Chinese alligator populations, in combination with 6 neutral microsatellite markers as a contrast. We found the genetic trace for the bottleneck effect on the endangered Chinese alligator: the low allelic diversity (2 alleles at each locus), the low nucleotide substitution rate (no more than 0.009) at all sites, the deviation from Hardy-Weinberg Equilibrium/heterozygote deficiency, and the significant Tajima’s D values, indicating the MHC class I and class II loci being at different stages of bottleneck. We also obtained 3 pieces of evidence for balancing selection on this severely bottlenecked reptile: an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions, the mean synonymous substitution rate of MHC exons significantly higher than mean nucleotide substitution rate of introns, and the differentiation coefficient F ST of MHC loci significantly lower than that of microsatellite loci. Consequently, we emphasize that the Chinese alligator holds a pretty low adaptive ability and requires scientific conservation strategies to ensure the long-term population development.