Buffalo is a high producing animal. But to exploit its full production potential is limited due to silent heat. Silent heat leads to improper diagnosis of estrus at the time of artificial insemination that causes low fertility in buffalo. Estrus is a quantitative polygenic trait controlled by environmental factors as well as polygenes. Among all the genes controlling estrus Leptin is the potential candidate gene for estrus trait and is positioned on chromosome 4q32. It stimulates production of GnRH and with FSH it controls production of estrogen thus affecting estrus behavior. The aim of the current study was to identify the single nucleotide polymorphisms in 5 flanking sequence of exon 1 and coding region of Leptin gene and to find their association with silent estrus trait. One hundred blood samples of Nili-Ravi breed were collected from UVAS Pattoki Campus, Research Farm B and Buffalo Research Institute (BRI) Pattoki. Inorganic method was used for DNA extraction and products were precipitated and sequenced for analysis. For the analysis of sequence and to identify the polymorphism bioinformatics software FinchTV software and Bioedit software were used. The 5 flanking sequence and total 3coding regions of Leptin gene were amplified with specially designed primers. The 15polymorphic sites were observed of which one SNP was found in intron 1,9 SNPs in exon 2, 4 SNPs in intron 3 and 1 SNP in exon 3 of Leptin gene. A Bioinformatics analysis was performed with the help of "POPGENE 32" software to find the association of identified polymorphisms with silent estrus. Four SNPs were found to have significant association with silent estrus with P<0.05. SNPs were analyzed for their effect and five SNPs in exon 2 were found to be synonymous, they changed the sequence of amino acids in the Leptin protein. Population genetic analysis and allelic distribution at all loci was analysed. Out of total fifteen polymorphisms, six haplotypes were constructed on the basis of DNA sequencing of individual samples. Statistical analysis of these haplotypes was done by using SHEsis software. SignalP software was used to predict the signal peptide of the Leptin protein. Phylogenetic analysis was performed and Parsimony trees were constructed by using Mega4 Software which showed sharing of cluster by Nili-Ravi buffalo breed and cattle. This genetic characterization of Leptin gene may serve as a powerful genetic source for the development of DNA markers that can be used in association studies and for selection of animals with good heat signs.