Lactoferrin (Lf), plays an important role in the innate immune system and defence mechanism of mammary gland of milk producing animals. The amount of lactoferrin increases during inflammatory process and viral infections. Many polymorphisms have been found in the bovine Lf Gene. The present study aimed to identify single-nucleotide polymorphic (SNP) site in the 5' untranslated region (5'-UTR, position +32) of the bovine Lf gene in Holstein dairy cattle and to quantify their association with clinical mastitis using Allele-specific polymerase chain reaction (PCR) method. Blood samples were collected from 89 multiparous Holstein dairy cows with a history clinical mastitis (n=38), and cows without mastitis as control group (n= 51). In this study, we detected type of SNP in the 5' untranslated region (5'-UTR, position +32) using a designed Allele specific PCR that validated following sequencing method. We did not found evidence for a significant association between SNP in the 5'-UTR (+32: G/G) Lf gene and the mastitis in dairy cattle in Iran. The results of this study did not provide any support to considered 5'-UTR polymorphism (position +32) as a marker for resistance/susceptibility to mastitis in dairy cattle.