The aim of the current research was to investigate the possible occurrence of a single nucleotide polymorphism (SNP) in exon 2 of the equine leptin gene in obese mares determined to be hyperleptinemic. Three experiments were conducted: one to determine the prevalence of hyperleptinemic horses in the resident herd; another to complete the sequencing of exon 2 and flanking introns of the equine leptin gene, which had been partially sequenced by others; and a third to compare the exon 2 sequences of obese, hyperleptinemic mares with those of obese mares not displaying hyperleptinemia. In experiment 1, jugular blood was collected from 31 mares, and they were categorized by age, body condition score (BCS), and average plasma leptin concentrations. Mean BCS was correlated (P < .001) with leptin concentrations; age was not. Five obese, hyperleptinemic and five obese, nonhyperleptinemic mares were selected to study the possibility of polymorphism in exon 2. First, in experiment 2, forward and reverse primers were designed from the Bos taurus leptin gene (GenBank Accession # U50365) to identify and subsequently clone the equine leptin gene to provide material for sequencing. The multiple copies of genomic DNA were then used for sequencing of exon 2 and the flanking introns. Comparative genomics was used to further identify and characterize regulatory elements in the 5' and 3' flanking regions of the leptin gene. In experiment 3, DNA from the five mares of each type (previously selected in experiment 1) was extracted, and exon 2 was sequenced and analyzed for possible SNP. The sequences of exon 2 for the 10 mares were identical; thus, no polymorphism was present. It was concluded that approximately one third of obese, pasture-maintained mares and geldings in the resident herd display hyperleptinemia relative to other horses of similar body condition, but this condition was not associated with the occurrence of SNP in exon 2 of the leptin gene.