Buffaloes’ productivity is affected mainly due to many inherent disorders. Estrogen hormone is found to be an important hormone directly linked with reproductive functions of buffalo. The present study was performed with 203 Murrah / Graded Murrah buffaloes maintained at different locations. The Polymerase Chain Reaction- Single Strand Conformation Polymorphism (PCR-SSCP) of ER-α gene revealed monomorphic pattern for all the tested animals, which indicates the fixation of allele in tested buffalo population. As a conclusion, monomorphic pattern of Estrogen Receptor-α (ERα) gene is considered a unique feature that may be related to the characteristic of buffaloes.