Genome analysis in animals has introduced new criteria for animal selection and strategies for increasing herd productive and reproductive potential through breeding. Leptin gene has established as a potential candidate for marker assisted selection of animals. The present study was aimed to investigate polymorphism in leptin gene of Nili-Ravi buffalo breeding bulls and its relationship with physical characteristics and reproductive traits for genotyping of breeding bulls. For this purpose, blood samples from 99 buffalo breeding bulls were collected for DNA extraction by using standard organic protocol. Primers for the amplification of leptin gene sequence were designed by using primer3 software (v. 0.4.0). Sequence comparison of leptin gene revealed, a total of 33 SNPs out of which 13 SNPs were found in the region of exon 2 and 3. While twenty (20) SNPs were found in intronic regions. To author’s knowledge three (3) SNPs at positions c.68, c.70 and c.158 in exon regions of leptin gene have been reported first time in buffalo. Analysis of variance and Pearson’s correlation was applied using R-statistical package (v. 2.15.2) for analysis of data. Results showed that the SNP at position c.20-91 had significant (p<0.01) positive correlation with dead sperm percentage, while it showed a negative correlation (p<0.05) with live weight of buffalo bulls, length from shoulder to pin-bone, individual sperm motility and live sperm percentage in the ejaculate. Leptin gene polymorphism at c.20-91 position significantly (p<0.01) affect ejaculatory volume and sperm motility after dilution. The single nucleotide polymorphism at position c.20-41 showed positive correlation (P<0.05) with girth, scrotal circumference and dead sperm percentage, while it was negatively correlated with sperm concentration and live sperm percentage. The c.20-27 polymorphism showed negative correlation (P<0.05) with age of buffalo bulls, live weight of bulls, length from shoulder to pin-bone, girth, scrotal circumference and testicular length of bulls. Effect of c.20-27 polymorphism on the distance between angles of the hip was also found significant (p<0.01) through analysis of variance (ANOVA). The polymorphism at position c.20-21 showed positive correlation (P<0.01) only with the live weight of buffalo bulls. The C>T change at position c.191+19 was positively correlated (P<0.05) with scrotal circumference and reaction time. Analysis of variance revealed significant effect of c.191+19 polymorphism on sperm motility after dilution. The SNP at position c.191+30 was found positively correlated (P<0.05) with body condition score and reaction time. Analysis of variance also verified its significant effect on the length from shoulder to pin-bone. The SNP at position c.191+41 revealed positive correlation (P<0.05) with live sperm percentage, while it was negatively correlated with dead sperm percentage. Similarly its effect on the distance between angles of the hip was significant (P<0.01) through analysis of variance. The genetic variant at position c.191+51 was positively correlated (P<0.05) with reaction time, while its correlation was negative with the live weight of buffalo bulls. Analysis of variance also showed significant (P<0.01) effect of c.191+51 SNP on the number of mounts per ejaculate. The c.191+99 SNP had a positive correlation (P<0.05) with live weight and live sperm percentage, while, it was negatively correlated with dead sperm percentage. Moreover, analysis of variance also confirmed its significant (P<0.01) effect on the number of mounts per ejaculate. The polymorphism at position c.284 was found to be negatively correlated (P<0.05) with length from shoulder to pin-bone and sperm motility after freezing. The c.422 polymorphism revealed significant positive correlation (P<0.05) with the height at withers, distance between angles of hip, scrotal circumference and testicular length. Analysis of variance also showed significant (P<0.05) effect of c.422 polymorphism on width of the face above eyes. However, the genetic variant at position c.523 showed significant negative correlation (P<0.05) with various parameters such as height at withers, length from shoulder to pin-bone and sperm motility after freezing. In conclusion, findings of present study suggests leptin gene to be a highly polymorphic with a significant correlation with different physical, reproductive performance and semen quality parameters in Nili-Ravi buffalo bulls. These SNPs can be considered as genetic markers.