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Background/Objectives: To establish a foundation for conserving and utilizing local frog germplasm resources in Zhejiang Province, for Quasipaa spinose, which has high commercial and nutritional value, a pan-genome analysis was performed. Methods: Herein, we characterized 405,263 SNPs for the giant spiny frog, Q. spinose, using the Illumina NovaSeq platform. Results: These loci were highly polymorphic in 59 individuals sampled from three different subpopulations, with 0.05 to 0.30 minor alleles per locus. The observed and expected heterozygosities were 0.2379 and 0.2683 (IBD), respectively. These polymorphic loci would be useful for assessing genetic diversity, population structure, gene flow, population assignment, and paternity in giant spiny frogs. Conclusions: Our investigation demonstrated that there are distinct genetic and evolutionary histories between Zhejiang and Jiangxi frogs. Phylogenetic inference effectively differentiated these three subpopulations based on their geographical origins, and the phylogenetic inference level of domesticated Zhejiang frogs was comparatively higher than that of the Jiangxi-derived population. Furthermore, by utilizing three selective signature methods, namely, Obs/Exp het, nucleotide diversity (Pi), and identical by state (IBS), across subpopulations, we concluded that these three breeds were from an identical population, and no genetic bottleneck occurred among these three lineages, in accordance with LD decay analysis. Finally, 2700 potential candidate genes were identified, including MAPK, calcium signaling pathway, Ras signaling pathway and regulation of actin cytoskeleton; we noted that the key genes associated with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy in humans beings and GnRH signaling pathway-related genes (i.e., CD80, IFNA, and KCNK1) were highly enriched, which could impact cardiac function through immune-associated genes.