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The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The aim of the present study was to detect the incidence of mutation in exon two of GDF9 as a major gene in the Shal sheep breed. Blood samples were collected from 239 sheep and genomic DNA was extracted using the modified salting-out method. The quantity and quality of extracted DNA was examined using spectrophotometery and gel electrophoresis, respectively. A fragment with the size of 139 bp from exon two of GDF9 gene (FecG H ) was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with DdeI restriction enzyme. In the presence of mutations at this locus, the DdeI enzyme cannot recognize the restriction site. However, in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 31 and 108 bp. In the present study only the wild type alleles were detected and all the samples showed the AA genotype. The analysis of polymorphism for GDF9 (FecG H ) loci in Shal sheep indicates that the genetic factor responsible for twinning or multiple lambing rates is not related to reported mutated alleles at the GDF9 major gene in this breed. Therefore, we should attempt to detect other SNP for the GDF9 gene and/or other loci responsible for twining rate in this breed. ________________________________________________________________________________