β-lactoglobulin is the major milk whey protein in ruminants and its coding gene located on ovine chromosome 3. This protein produces in mammary glands during pregnancy and lactation stages. Studies have shown that the protein is polymorphic in many breeds of sheep. This is the result of single base pair substitution in β-lactoglobulin gene that also rises to RsaI restriction Fragment Length Polymorphism (RFLP). Blood samples were supplied from 142 animals of Iranian sheep breeds (Ghezel, Afshary, Moghani, Makoii and Arkharmerino). Genomic DNA was extracted from 100 μL blood sample according to Boom method modified by Shaikhayev. Gel monitoring and spectrophotometeric methods were used for determination of DNA quality and quantity. BLg 5 and 3 primers amplified a 452 bp fragment from exon II of ovine β-lactoglobulin gene. Products of amplification were recognized by electrophoresis on 1% agarose gel stained with ethidium bromide. RsaI enzyme was used for restriction of PCR products. Digestion products were separated by electrophoresis on 8% non-denaturant polyacrylamide gel and visualized after staining with ethidium bromide on UV gel documentation. PopGen 32 software (ver. 1.31) was used to estimating the frequency of allele, genotype and Hardy-Weinberg equilibrium. Frequency of A-allele in Ghezel, Afshary, Moghani, Makoii and Arkharmerino sheep was 56, 34, 36, 53 and 48%, respectively. The populations were in Hardy-Weinberg equilibrium except of Afshary breed.

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