The objective of the work is to improve the twinning rate in Rahmani sheep (R) local breed through crosses with prolific sheep breeds; Finn (F) and Romanov (Rv) and to study the gene of twinning in the crossbreds using Growth Differentiation Factor 9 (GDF9). Blood samples were collected from the studied genotypes (R, F, Rv, ½ FR, ½ RvR, ¼ FR, and ¼ RvR) (73 ewes) to extract DNA. A specific GDF9 gene was amplified for exon1 and exon2. Several polymorphisms have been identified for exon2. The comparative alignment for sequencing of Rahmani, Finn, Romanov, ½ Finn, and ½ Romanov showed 20, 7, 23, 9, and 11 variations, respectively, where the highest was in the Romanov. However, variations in reverse sequences were high in Rahmani sheep and low between Finn, Romanov, and their crossbreds. Twenty-nine mutants were detected, five of them were two single-nucleotide-polymorphisms and three-gaps that can be used to predict the prolificacy of young crossbred lambs with accuracy and differentiate between single and multiple births.