In recent years the number of malignant mammary gland tumor occurrences in domestic bitches has increased. This may be related to advanced diagnostic technologies, availability of a quick diagnosis, as well as genomic changes. Still the main genetic reasons for tumor development are frequently occurring gene mutations and/or polymorphisms. The mutations in target genes often lead to amino acid changes in the structure of proteins, which may be the reason for uncontrolled cells proliferation, and induction and growth. Therefore, in this article we described the analysis of mutation/polymorphisms frequency of the GGA1 gene in association with canine malignant mammary gland tumor occurrence and aging. In this study, blood samples were obtained from 22 female dogs diagnosed with mammary tumors. Moreover, blood samples from geriatric (>5 to 10 years old; n = 15), mature adult (> 2 to 5 years old; n = 10) and young (from 1 to 2 years old; n = 11) dogs were also collected. 36 bitches diagnosed on account of other reasons served as controls. After the Sanger sequencing analysis, 14 single nucleotide variations were identified, of which 3 were already known polymorphisms and 11 novel variations. We observed differences in frequencies for 4 polymorphisms (g.A-172T, c.T24C, c.A692G, c.C1185T) between cases and controls. Moreover, we found increased prevalence of heterozygotes and alternative alleles in 3 polymorphisms (g.A-172T, c.A692G, c.C1185T) in the tumor diagnosed group as compared to the control. Although the results were not statistically significant, it is worth mentioning the slightly different pattern of genetic segregation of alleles in the abovementioned polymorphisms in control and tumor patients.