Background: The palate is a structure separating the oral and nasal cavities and its integrity is essential for feeding and breathing. The total or partial opening of the palate is called a cleft palate and is a common malformation in mammals with environmental or hereditary aetiologies. Generally, it compromises life expectancy in the absence of surgical repair. A new form of non-syndromic cleft palate arose recently in Limousine cattle, with animals referred to the French National Observatory of Bovine Abnormalities since 2012. Since the number of affected animals has increased steadily, this study was undertaken to identify the cause of this disease. Results: Based on pedigree analysis, occurrence of cleft palate in Limousine cattle was concordant with an autosomal recessive mode of inheritance. Genotyping of 16 affected animals and homozygosity mapping led to the identification of a single disease-associated haplotype on Bos taurus chromosome (BTA)19. The genome of two affected animals was sequenced, and their sequences were compared to the ARS-UCD1.2 reference genome to identify variants. The likely causal variants were compared to the variant database of the 1000 bull genome project and two fully linked mutations in exon 24 of the MYH3 ( myosin heavy chain ) gene were detected: a 1-bp non-synonymous substitution (BTA19:g.29609623A>G) and a 11-bp frameshift deletion (BTA19:g.29609605-29609615del). These two mutations were specific to the Limousine breed, with an estimated allele frequency of 2.4% and are predicted to be deleterious. The frameshift leads to a premature termination codon. Accordingly, mRNA and protein analyses in muscles from wild-type and affected animals revealed a decrease in MYH3 expression in affected animals, probably due to mRNA decay, as well as an absence of the MYH3 protein in these animals. MYH3 is mostly expressed in muscles, including craniofacial muscles, during embryogenesis, and its absence may impair palate formation. Conclusions: We describe a new form of hereditary cleft palate in Limousine cattle. We identified two fully linked and deleterious mutations, ultimately leading to the loss-of-function of the MYH3 protein. The mutations were included on the Illumina EuroG10k v8 and EuroGMD v1 SNP chips and are used to set up a reliable eradication strategy in the French Limousine breed.