The Prophet of Pit1 (PROP1) gene isoforms are reported to be associated with production, growth and fertility trait in cattle. The present study was carried out to screen the Sahiwal bulls for the PROP1 gene isoforms resulting from H173R mutations in third exon of the gene. The exon 3 was characterized by sequencing the amplicons obtained after PCR amplification using custom designed primers. Phylogram of exon 3 of PROP1 identified that Sahiwal is closely related to Bos taurus and Karan Fries in evolutionary tree whereas it is distantly placed from rodents. The multiple alignment of the target region sequence with Bos taurus reference sequence revealed that the bulls under the study were free of mutations causing the PROP1-173R isoforms. No other variations were observed in the third exon thus giving the targeted region a highly conserved one.