Abstract: The part of the bovine genome which shows a superior action and explains the major part of variation of the economical production traits were known as QTL. PIT1 gene which is also termed hormone factor-1 is a pituitary-specific transcription factor which has responsible for pituitary development and hormone expression in mammals. The main function of PIT1 is for binding and trans-activity promoters of both Growth Hormone (GH) and Prolactin (PRL) gene. It was subjected to different molecular studies as a key role for understanding genetic variation in dairy cattle. This study was aimed to detect the polymorphism in a part of PIT1 gene, exon 6 in Najdi cattle. DNA was extracted from blood sample collected from 84 Najdi Cattle and submitted for Polymerase Chain Reaction (PCR) followed by digestion with HinfI restriction enzyme. Base on PCR-RFLP method, it found that the frequency of the A and B alleles of this gene for Najdi Cattle were 18.45 and 81.55%, respectively. The genotypic frequencies of AA, AB and BB were 3.57, 29.76 and 66.66%, respectively. χ2-test indicated that these two populations were at Hardy-Weinberg equilibrium. Thus, the selection base on these loci in the population is possible to be done.