Leptin is a 16-kDa polypeptide hormone synthesized and secreted predominantly by adipose tissue. It functions to regulate body weight, food intake, energy expenditure, reproductive and immune functions. Leptin treatment of animals has been shown to cause a decrease in food intake, body weight loss, fat deposit weight loss and increase in energy metabolism. Therefore leptin not only causes reduced food intake, but the potential body weight losses are enhanced due to an increased metabolic rate (Houseknecht et al. 1998). The gene encoding Leptin was mapped to Bovine chromosome 4 and it consists of 3 exons and 2 introns of which only two exons are translated into protein. The coding region of the leptin gene (501 nucleotides in length) is contained in exon 2 and 3, which are separated by intron of approximately 2 kb. Leptin is considered as a candidate gene for milk performance related traits in cattle. Several polymorphism in this gene have been found (Liefers et al. 2002). In exon three, A59V polymorphism, causes as amino acid change from Alanine to valine. These amino acids both belong to the group of aliphatic amino acids, but Valine is more hydrophobic. The aim of this study was to analyse the frequency of the A59V Polymorphism in exon 3 of Leptin Gene in Iranian Holstein cattle.