Litter size is one of the most important reproductive criteria in animals. Hence, the effect of this trait on the productivity of the flock has been addressed by its genetic enhancement through molecular technologies. Previous studies have shown that the major genes including GDF9, BMP15, ALK6, and B4GALNT2 (Fec^L) are associated with litter size. The effect of Fec^L mutation in homozygous states is additive and results in the release of three ovum for two copies of this gene. The aim of this study was to investigate polymorphism DLX3: c.­* 803A> G in B4GALNT2 (Fec^L) gene on chromosome 11, using High Resolution Melting technique (HRM), and it is association with litter size in Zandi sheep. HRM analysis is a method that reduces the amount of fluorescence during the process of thermal melting of the DNA through color exhaust. The results of HRM were consistent with the sequencing results. No mutations were observed in the nucleotide sequence of the target studied in the Zandi sheep. Therefore, B4GALNT2 gene is monomorphic for this site length in 232bp fragment of this gene. We discovered many deletions, but there was no change in the resulting amino acid. Therefore, that did not result in structural alteration of the protein. In addition, two new mutations included A> G mutation at position 2242 in sample 7 and mutation C> G at position 2241 in sample 77 were detected. It seems necessary to investigate mutations in other major genes for this breed