Abstract Several polymorphisms in the coding region of the myostatin (MSTN) gene have been associated with the double-muscled (DM) phenotype in different species. An experimental herd of purebred Raramuri criollo cattle in Chihuahua, Mexico, has been selectively managed for beef production during the last 15 yr. Recently, two calves where visually identified as DM animals. The aim of this study was to identify changes in the MSTN gene sequence, associated with the two DM individuals and to screen for carriers of the potential polymorphisms within this herd. The DM paternity of the calves was verified by the SeekSire bovine parentage testing of NEOGEN (Lincoln, NE). A 6.6 Kb fragment of the MSTN gene were amplified by PCR and sequenced using Illumina DNA prep kit in a MiniSeq platform. The resulting reads were analyzed using different bioinformatic programs. SNPs were generated in VCF files and filtered by the following criteria: variant confidence normalized by depth of variant samples (QD) < 2.0, mapping quality (MQ) < 40.0, strand bias(FS) > 60.0. The deduced myostatin protein and the three-dimensional model were obtained with bioinformatic tools. Once a novel variant was identified, a real-time PCR protocol was developed to genotype 100 and 97 herdmates, the allelic and genotypic frequencies were estimated. A 9-bp deletion was identified at the same position as the 11-bp deletion (g.821to 831del11) of the Belgian Blue MSTN allele (Figure 1). Although the latter allele has been found in three Spanish cattle breeds ancestrally related to Raramuri criollo, this is the first report of the 9-bp deletion in the MSTN gene. Such deletion results in the loss of three highly conserved amino acids residues (274, 275 and 276) in MSTN, but does not lead to a premature stop codon as in the case of the 11-bp deletion characteristic to DM Belgian Blue. The in silico three-dimensional MSTN conformation obtained for this allele has some structural differences compared with the protein reference model. The allelic frequencies were 0.85 and 0.15 for the MSTN+ and MSTNdel alleles, respectively, whereas the genotypic frequencies were 0.71, 0.01 and 0.28 for MSTN+/MSTN+, MSTNdel/MSTNdel MSTN+/MSTNdel genotypes, respectively. Controlled mattings to generate different genotypes in the research herd would allow to continue studying the effects of this MSTN gene variant on productive performance, forage behavior, husbandry and carcass and meat quality traits known to be affected by the DM phenotype in beef cattle.