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Since lactoferrin is acute phase protein and has an exceptional role in defense mechanism of mammary gland, it is considered a candidate gene for mastitis susceptibility in dairy cattle. In this study, blood samples were collected for DNA extraction from fifty Holstein dairy cows in the third lactation season reared under Egyptian conditions assigned into three groups mastitis (n=15), subclinical mastitis (n=20) and healthy (n=15) based on California mastitis test. Moreover biochemical markers for inflammation were determined to detect severity. PCR amplification of a segment of 6th intron of lactoferrin gene yielded a fragment of 301-bp in all animals under study. For revealing polymorphism, DNA sequencing was done for PCR products of lactoferrin gene (301-bp) in only five healthy (resistant) and five affected animals. Results indicated that, association between biochemical data and affections. The PCR-DNA sequencing genetic assessment identified twelve SNPs in the bovine lactoferrin gene and there was association between these identified SNPs and mastitis susceptibility, where ten nucleotide sequence variations for one of the healthy animals were obtained with A166T, T185A, T200C, G230C, A239G, T251G, A254C, A272C, T273C and A296G SNPs (submitted to GenBank with accession number gb|KT159457|. On the other hand, two nucleotide sequence variations for two of the affected animals were obtained with A100G and T275A SNPs. Consequently, the identified SNPs in the bovine lactoferrin gene can be used as marker-assisted selection (MAS) to predict, improve mastitis resistance and minimize incidence of mastitis infection in Holstein dairy cows.