Buffalo are shy breeders and poor fertility traits are a major hindrance in exploiting the production potential of the animal. This study hypothesizes that polymorphisms in the luteinizing hormone beta (LHβ) gene can affect oestrus behaviour in buffaloes. A total of 100 animals were screened by calculating the heat index (threshold-50) and animals were categorized into two groups (Group1 > 50, Group2 < 50). Animals were subjected to blood sampling, genomic DNA isolation, specific primer based polymerization and sequencing of amplicons. A total of six genomic variations were identified in the gene. c.V15M was a non synonymous mutation found in line with the Hardy Weinberg Equilibrium and was significantly associated with the trait. Functional impact of the variation was determined by three-dimensional structure of the protein. Effect of c.V15M on the functionality of the gene was evident and hypothesis was supported so this can potentially be used as a marker for the future development of superior animal breed or regulating the expression of the gene to get the optimal oestrus cyclicity in river buffalo of Pakistan.