Pit-Oct-Unc Domain, Class 1, Transcription Factor 1 (Pit1)is a member of the tissue-speciﬁc POU homeobox transcription factor family that is found in all mammals. Pit1 encodes a pituitary-specific transcription factor, which is involved in pituitary development and regulating the hormone expression in animals. In current study, Pit1 gene was screened for polymorphic sites in Sahiwal cattle. Samples from Sahiwal cattle breeds were sequenced using six set of primers. A total 15 polymorphisms; 12 in intronic and 3 in exonic region, were identified. The sequences of the amplified Pit1 gene fragments were aligned with the help of BLAST for SNPs identification. This is a first report toward genetic screening of this gene at molecular level in Sahiwal cattle of Pakistan. No work has been reported on this gene in Sahiwal cattle. In this study, a new set of single nucleotide polymorphisms (SNPs) were reported. Heterozygosity, allelic and genotypic frequencies of identified variants were also determined. Chi2 test was performed to evaluate the Hardy-Weinberg Equilibrium of each polymorphic site. Two loci were found to be in HWE. These identified alleles will be useful for animal selection at molecular level i.e. an ideal tool for marker-assisted selection of animals for future breeding programs.