Tumorigenesis is associated with metabolic abnormalities and genomic instability. Microsatellite mutations, including microsatellite instability (MSI) and loss of heterozygosity (LOH), are associated with the functional impairment of some tumor-related genes. To investigate the role of MSI and LOH in sporadic breast tumors in canines, 22 tumors DNA samples and their adjacent normal tissues were evaluated using polyacrylamide gel electrophoresis and silver staining for 58 microsatellites. Quantitative real-time polymerase chain reaction, promoter methylation analysis and immunohistochemical staining were used to quantify gene expression. The results revealed that a total of 14 tumors (6 benign tumors and 8 breast cancers) exhibited instability as MSI-Low tumors. Most of the microsatellite loci possessed a single occurrence of mutations. The maximum number of MSI mutations on loci was observed in tumors with a lower degree of differentiation. Among the unstable markers, FH2060 (4/22), ABCC9tetra (4/22) and SCN11A (6/22) were high-frequency mutation sites, whereas FH2060 was a high-frequency LOH site (4/22). The ABCC9tetra locus was mutated only in cancerous tissue, although it was excluded by transcription. The corresponding genes and proteins were significantly downregulated in malignant tissues, particularly in tumors with MSI. Furthermore, the promoter methylation results of the adenosine triphosphate binding cassette subfamily C member 9 (ABCC9) showed that there was a high level of methylation in breast tissues, but only one case showed a significant elevation compared with the control. In conclusion, MSI-Low or MSI-Stable is characteristic of most sporadic mammary tumors. Genes associated with tumorigenesis are more likely to develop MSI. ABCC9 protein and transcription abnormalities may be associated with ABCC9tetra instability.