MyoD1 gene is a member of the myogenic differentiation gene family, which plays a crucial role in growth and development. There is a lack of information about the MyoD1 variants and their phenotypic influences in Turkish Grey Steppe cattle. Therefore the present study aimed to determine the genetic variability of MyoD1 g.782G>A polymorphism and to evaluate its effects on carcass characteristics in Turkish Grey Steppe cattle. Analyses were conducted on a total of 142 Turkish Grey Steppe bulls. The PCR-RFLP technique was used for genotyping of the g.782G>A polymorphism in exon 1 of the MyoD1 gene. The population genetic parameters including compatibility with Hardy-Weinberg equilibrium, heterozygosity, number of effective alleles, the polymorphism information content, and the fixation index were calculated. Statistical analysis was carried out using the least square methods of the general linear model procedure. Results revealed that BB was the preponderant genotype (41.55%) but the AA genotype exhibited a remarkable close frequency (39.44%). Accordingly, allelic frequencies were very close to each other (A:0.49; B:0.51). The chi 2 test revealed that the corresponding MyoD1 locus did not conform to Hardy-Weinberg equilibrium (p < 0.001). Heterozygosity, number of effective alleles, and the polymorphism information content values were 0.4998, 1.9992, and 0.3749, respectively. Moreover, the fixation index value was found to be 0.6195. These results indicated that MyoD1 g.782G>A polymorphism is a mildly informative genetic marker for Turkish Grey Steppe cattle. Statistical analysis indicated that the studied MyoD1 locus was significantly associated with chilling loss, carcass bone content, and carcass length (p < 0.05). The AA genotype was characterized by a higher chilling loss percentage but lower carcass length and bone content compared to BB and heterozygous genotypes. This study may provide valuable information regarding cattle carcass assessment and improvement through marker-assisted selection.