Prolactin (PRL) hormone plays an important role in many biological processes in animals and humans, such as mammogenesis, lactogenesis and galactopoiesis. PRL is a single-chain protein of 199 amino acids  (AA) in human, sheep, pigs and cows. The objective of the present study was to determine the polymorphism in exon 4 of PRL gene in 41 Najdi and 36 Naeimi sheep breeds of Saudi Arabia. A fragment of 156 bp of exon 4 of the Prolactin gene was amplified  and sequenced. Our  mutations were detected at positions (05, 109 and 127) of exon 4. In addition, the three mutations were used to construct different haplotypes, four haplotypes were recovered, H1 (CGG), H2 (CGT), H3 (AGG) and H4 (CCG). The H1 (CGG) was the most common haplotype and found in Najdi and Naeimi individuals with frequencies of 0.927% and 1.00% for Najdi and Naeimi sheep breeds, respectively. The haplotypes  H2, H3 and H4 were unique for Najdi sheep with frequency of 0.025% for each. Naeimi sheep breed was monomorphic at exon 4 of the Prolactin gene. The present study provides basic information to understand the genetic Characterization of local sheep breeds in Saudi Arabia.