Extracellular superoxide dismutase (SOD3) is a major antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). In this study, the SOD3 gene was identified and characterized from the freshwater mussel Hyriopsis cumingii (Hc-SOD3). The cDNA sequence consists of 763 bp, encoding a protein of 208 amino acids. The amino acid sequence possesses two CuZnSOD signature sequences, and amino acids required for binding of Cu (His-93, -95, -110 and -169) and Zn (His-110, -118, -129 and Asp-132) were conserved in Hc-SOD3. The Hc-SOD3 genomic sequence was 9165 bp in length, containing four exons and three introns. Eighteen single nucleotide polymorphisms were detected in the Hc-SOD3 gene from resistant stock (RS) and susceptible stock (SS) of H. cumingii to Aeromonas hydrophila. The genotype and allele distribution were examined in resistant and susceptible stocks. Among them, a C/G substitution at the g.7994C>G locus and G/C substitution at the g.8087G>C locus were significantly associated with resistance/susceptibility of H. cumingii to A. hydrophila, both in genotype (P = 0.017, P = 0.004 respectively) and allele frequency (P = 0.021, P = 0.006 respectively). Linkage disequilibrium analysis revealed that g.7994C>G, g.8001A>G, g.8035G>A, g.8087G>C and g.8191T>A were in linkage disequilibrium. The results suggest that the two polymorphic loci, g.7994C>G and g.8087G>C, could be potential genetic markers for future molecular selection of strains that are resistant to diseases.