Hypertrophic cardiomyopathy (HCM) is one of the most common heart diseases among cats. The aim of the study was to find out the prevalence of the missense A31P mutation of the MYBPC3 gene in the feline population and its dependence on breed and sex. During the study, 130 individuals were tested by PCR-RFLP method. The 242 bp PCR product was digested with AvaI restriction enzyme and the fragments were separated by electrophoresis in 2.5% agarose gel. Besides that, retrospective analysis was performed based on data from the veterinary clinic, collected in a three years period. The mutation (G/C genotype) was detected only in the Maine Coon breed. Data analysis showed that frequency of heterozygous genotype in the tested Main Coon population was 0.23, which is equal to 13.85% of all the studied feline population and 23.1% of the tested experimental (Maine Coon) group. Allele frequencies were calculated in the experimental group (Maine Coon), the control group (feline of other breeds) and for all tested individuals. In all groups, the most frequently repeated allele was G, and in the control group, it was the only detectable allele. Frequency of the C (mutated) allele was calculated for all individuals (0.07) and for experimental (Maine coon) (0.115) groups. Statistically significant Main Coon breed dependence on the mutation was assessed (P < 0.05). Information analyzed retrospectively showed that HCM was more often diagnosed for males (87%) than females.