Phosphoenolpyruvate carboxylase is a key enzyme in the gluconeogenesis pathway. When animals and plants are under adverse environmental conditions such as temperature stress, they can convert oxaloacetic acid into phosphoenolpyruvate, generating glucose to maintain the blood glucose balance. This process plays an important role in stress resistance. In this study, PEPCK gene of Pinctada fucata martensii was identified, the changes of PmPEPCK expression under low temperature stress were analyzed, and single-nucleotide polymorphism (SNP) sites in the exon region of PmPEPCK in low temperature tolerant breeding line F3 (R) and Beibu Gulf wild populations (W) of Pinctada fucata martensii were screened and compared. Sequence analysis showed that the PmPEPCK gene had a full length of 2557 bp; the 5?UTR and 3?UTR were 56 bp and 446 bp long, respectively, and the open reading frame was 2055 bp long and encoded 684 amino acids. PmPEPCK was expressed in the tested tissues, among which the hepatopancreas had the highest expression (P＜0.05). The time-series expression results in gill tissues after low temperature stress showed that the expression of PmPEPCK in the low temperature group reached the highest at 12 ℃ and 17 ℃ at 6 h, which was significantly higher than that at 22 ℃ noted in the control group (P＜0.05), indicating that PmPEPCK participates in the low temperature response process of shellfish. A total of 108 SNPs were identified in the exon region of PmPEPCK, of which 47 SNPs showed significant differences between the R and W population (P＜0.05). Among the 47 SNP loci, 11 genotypes only appeared in R, which were the AG genotype of loci g.60010420, g.60007411, and g.60011365; genotype AA of loci g.60006265 and g.60006166; genotype CT of loci g.60007389 and g.60011387; genotype TT of loci g.60009449 and g.60006028; genotype GG of locus g.60011341; and genotype GT of locus g.60009464. Thus, after 3 generations of selection, the PmPEPCK gene of the R population was strongly positively selected. These SNPs and their genotypes of PmPEPCK may be related to the low temperature tolerance of the breeding line and used as candidate markers for selective breeding.