Buffalo is the main source of white milk and contributes 45% of the annual milk production in Egypt. The STAT5A protein has an important role in the signal transduction process within uterine epithelial cells and mammary glands affecting milk yield, fertilization, and embryonic survival rates in cattle. The studies conducted for the improvement of the genetic potentiality of productive and reproductive traits of Egyptian buffalo are still scanty. Data on polymorphisms of the buffaloes STAT5A gene is limited. Hence, the present study aimed to identify novel polymorphisms in the STAT5A gene and to validate its genotypic frequencies in Egyptian buffalo. A 929 bp including part of intron 7 to part of intron 9 and the intervening exons of the STAT5A gene was amplified and genotyped by the SSCP method. Three different conformation patterns in the investigated 60 buffaloes were observed. Pattern B showed the highest observed frequency of 51.7%, while patterns A and C occurred at frequencies of 45% and 3.3% respectively. A Chi-square test showed that the Egyptian water buffalo population was in Hardy-Weinberg equilibrium. STAT5A gene might be utilized as a potential molecular marker for effective animal selection and breeding programs.