Canine degenerative myelopathy (CDM) is a spontaneous neurodegenerative disease. Genetically, CDM is an autosomal recessive disease with incomplete penetrance, most commonly caused by a genetic mutation in exon 2 of gene SOD1 (c.118G > A). This study aimed to determine the mutant allele frequency associated with CDM in various dog breeds from Romania. Dogs (n = 230) from 26 breeds were included in the study. Genotyping using the PCR-RFLP technique was performed on DNA extracted from oral swabs. The results revealed that 204 dogs were homozygous for the wild-type allele (G/G), 16 were heterozygous (A/G), and 10 were homozygous for the mutant allele (A/A). The mutant allele was identified in Wire Fox Terrier, Romanian Mioritic Shepherd, German Shepherd, Rottweiler, Belgian Shepherd, and Czechoslovakian Wolfdog breeds. The mutant allele frequency (A) within the tested population was 0.0783. The results for Belgian Shepherd, German Shepherd, and Romanian Mioritic Shepherd were in Hardy–Weinberg equilibrium, but a departure was observed for Rottweiler. The current study included a first screening of the Romanian Bucovina Shepherd, Romanian Mioritic Shepherd, and Caucasian Shepherd breeds. Genetic testing for the mutation associated with CDM is important in order to avoid the risk of the emergence of dogs homozygous for the SOD1:c118G > A allele.