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Brazil is one of the world's largest beef exporters, although the product has a low price due to quality issues. The meat exported by Brazil is considered medium and low quality by international buyers, mainly due to lack of tenderness. The predominant Zebu breeds (80% Nellore) are known for producing tougher beef than taurine breeds. Nonetheless, some studies have shown that there is substantial genetic variability for tenderness within the Nellore breed, although it is a difficult trait to improve by conventional selection methods. Therefore, the aim of this study was to perform a genomewide association study (GWAS) and a gene set enrichment analysis to identify genomic regions and biologically relevant pathways associated with meat tenderness in Polled Nellore cattle. Data consisted of Warner-Bratzler shear force values of LM from 427 Polled Nellore animals divided into 3 experimental slaughters (years 2005, 2008, and 2010). The animals were genotyped with either the Illumina BovineHD BeadChip (777k, on 61 samples) or the GGP Indicus HD chip (77k, on 366 samples). Single nucleotide polymorphisms were excluded when the call rate was <90%, the Hardy-Weinberg proportions -value was <1% (Fisher exact test, Bonferroni adjusted), and the minor allele frequency was <1%. Imputation from the GGP Indicus HD chip to the Illumina BovineHD BeadChip was performed using the FImput program. Genomewide association analysis was performed using the Efficient Mixed Model Association eXpedited (EMMAx) and the population parameters previously determined (P3D) methods. The GWAS was complemented with a gene set enrichment analysis performed using the FatiGO procedure. Significant markers ( < 0.0001) explaining a larger proportion of variation than other significant SNPs were located on chromosomes 3, 13, 17, 20, 21, and 25, indicating QTL associated with meat tenderness throughout the genome. Additionally, gene set analysis identified 22 Gene Ontology functional terms and 2 InterPro entries that showed significant enrichment of genes associated with tenderness. The functional categories included protein tyrosine and serine/threonine kinase activity, calcium ion binding, lipid metabolic process, and growth factors, among others. These results help to elucidate the genetic architecture and metabolic pathways underlying this trait, which is of extreme economic and social importance to Brazil, because Nellore is the dominant beef cattle breed in the country.