SPEF2 gene represents one of the important genes that are directly related to the fertility of farm animals, because of its work in the production of a protein that participates in the normal growth of the tail of the sperm and thus has a direct effect on the motility of the sperm. This research seeks to uncover the haplotype of the SPEF2 gene in both Holstein Iraqi bulls and male Iraqi buffaloes. 7 bulls and 7 male buffaloes were used, belonging to the artificial insemination centre in Abu Ghraib (fertility information) between the ages of 2-3 years for the period from 10/11/2022 to 10/2/2023. All animals were under veterinary supervision. By using a synthetic vagina, semen had been collected, then DNA was obtained, PCR amplification was performed, after that, after purifying the product of PCR, the sequence analysis was performed. Some bioinformatics analyzes were conducted to detect possible haplotypes of SPEF2 gene in each of buffalo cattle. Two haplotypes were obtained in each of the cattle and buffaloes, this diversity in haplotypes can explain the genetic diversity of this gene in farm animals in Iraq and can contribute to the selection of animals with high fertility, as this gene can be considered as a molecular marker for the selection of fertile males.