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Background: Pyometra, involves pus accumulation that can lead to death if detected late, making early diagnosis and management crucial, and it is more common in older dogs. The Axiom™ Canine HD Array was used in the study to determine single nucleotide polymorphisms (SNPs) associated with pyometra, with the aim of providing foundational data for the future development of SNP markers. Methods: Samples were collected from 95 dogs of 26 breeds in South Korea, and SNP genotypes were analyzed for the final two SNPs (AX-168186923, AX-168208364). To verify the sequences and genotypes of the selected SNPs, we conducted a sequencing analysis of 96 dogs from 11 breeds previously reported to have been affected by the disease. To confirm the linkage disequilibrium (LD) between the selected SNPs and the influence of non-synonymous SNP (nsSNP), candidate gene function and, protein structure change prediction analyses were performed. Results: Sequence and genotype analyses of the selected SNPs revealed differences in genotype frequencies among the varieties. A strong LD block was formed between the SNPs, confirming that both SNPs were genetically associated. Candidate gene ABCC4 analysis revealed that ABCC4 transports prostaglandins. An analysis of the three-dimensional protein structure affected by the nsSNP (AX-168186923) showed that the amino acid changed from Methionine to Valine, but the overall protein structure remained unchanged. Conclusions: The SNPs selected in this study can be used to identify genetic factors, associated with pyometra and vulnerable breeds. This is expected to reduce companion animal related costs and the psychological burden on owners.