Knowledge about the 5′ regulatory region variation of candidate genes may help to improve breeding and preserve genetic diversity in ruminant species and reveal insights about their evolutionary history. Cis-regulatory regions of candidate genes involved in lipid metabolism and related to energy homeostasis were sequenced in 3 Bos taurus breeds and 1 Bos indicus breed to identify potentially functional polymorphism in non-coding regions. Searching for promoter variability in Fatty Acid Synthase (FASN), Stearoyl-CoADesaturase (SCD), Perilipin (PLIN), Glycerol-3 phosphate acyltransferase mitochondrial (GPAM) and Melanocortin-4 receptor (MC4R), 42 polymorphisms were found. Forty one of them were reported for the first time in B. taurus and B. indicus species. Through in silico analysis we noticed that many of them fall within putative binding sites for transcription factors closely involved in the regulation of the expression of adipogenic genes. A higher nucleotide diversity was found in B. indicus for FASN, SCD, PLIN and MC4R, whereas GPAM was the only more polymorphic promoter in B. taurus. The lower diversity in the B. taurus breeds may reflect their population history with a stronger selection pressure for some economic and productive traits. Alternatively, the ancestors of the indicine breed may have had a high genetic diversity. This research underlines the significance of local breeds for preserving genetic diversity for a sustainable agriculture. The domestication history of ruminant species and the critical role of the analyzed genes in lipid metabolic processes, make these putatively regulatory polymorphisms important targets in breeding and in genetic diversity studies.