The aim of this study was to use the RNaseq technique to identify differentially expressed (DE) genes in the Longissimus thoracis muscle of Nellore cattle finished in feedlot with extreme phenotypes for beef fatty acid profile. After slaughter, a muscle tissue sample was collected of each animal for the extraction of RNA and in the deboning a sample was taken for determining the fatty acid profile. The fatty acids were quantified by gas chromatography (CG-2010 Plus; Shimadzu), using capillary column SP-2560. The following fatty acids were quantified: myristic (C14:0), palmitic (C16:0), stearic (C18:0), oleic (C18:1 cis-9), linoleic (C18:2 cis-cis-9–12), CLA (C18:2 cis-9 trans-11) and linolenic (C18:3), total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), total polyunsaturated fatty acids (PUFA), the ratio of polyunsaturated fatty acids on saturated (PUFA/SFA), n-3 (omega-3) fatty acids and n-6 and ratio of n-6 fatty acids on n-3 (n-6/n-3). Two groups of animals with extreme phenotypes for the composition of meat fatty acid were formed, being considered 10 animals with the highest (H) and 10 animals with the lowest (L) concentrations for each fatty acid. The RNA sequencing data were generated on the Illumina HiSeq System platform. The differential expression of RNA was determined using the iPlant Collaborative platform, containing the FastQC package (version 0.10.1), TopHat2 (version 2.0.9) and Cuffdiff (2.1.1). The analysis of the metabolic pathways of differentially expressed genes was performed with the DAVID tool. The ACSS1 was upregulated (q < 0.05) for saturated fatty acids (palmitic, stearic, oleic, total saturated), and downregulated (q < 0.05) for unsaturated acids, (omega-3). This gene assists in the conversion of acetate, into acetyl-CoA to incorporate it into the fatty acids by the action of acetyl CoA synthetase. Other DE genes involved in metabolic pathways of fatty acids synthesis were found, such as: BDH1_BOVIN, ACSM3, CBPE_BOVIN, F1N650_BOVIN. Through the RNaseq technique was identified possible genes acting in the synthesis of beef fatty acids, which can be beneficial for human health.