Cow milk contains approximately 3.5% protein, which make up 80% caseins and 20% whey proteins. The serum (whey protein) consists of approximately 50% ?-lactoglobulin, 20% ?-lactalbumin, blood serum albumin, immunoglobulins, lactoferrin, transferrin, and many minor proteins and enzymes. There is little or no evidence to present phosphorus and sulfur-containing amino acids in this part of milk. Bovine lactoferrin (LTF), a multifunctional small glycoprotein, acts as a defense factor in the mammary gland. It is synthesized by granulocytes and mammary epithelial cells in response to the infection. This protein is an iron-binding 80-kDa glycoprotein initially found in milk and in other fluids like saliva, tears, yellow bile, mucous secretions and blood. The bovine LTF gene is located on the 22nd chromosome and consists of 17 exons, 1122 bp of promoter region and spans about 34.5 kb of the genomic DNA. The objectives of this study were to estimate the allelic and genotypic frequencies of lactoferrin gene in Holstein cows and also evaluating the association between gene polymorphism and the milk and reproductive traits. For this purpose, blood samples were collected from 404 cows of five Holstein dairy herds in Isfahan province. Number of Samples varied from 64 to 96 among the herds. DNA was extracted using salting out procedure. The DNA quality and quantity were determined using electrophoresis and spectophotometry. The 301 bp fragment of the gene was amplified by specific primers and Thermocycler. PCR products were detected by electrophoresis on 1% TAE agarose gel. Amplified fragment digested via EcoRI restriction enzyme in 8 to 14 hours at 37 ? C. The genotype Frequencies of AA and AB were 0.606 and 0.394, respectively. In the all herds, BB genotype not observed. Allele A occurred at higher allelic frequency than allele B. The frequencies of alleles A and B were 0.806 and 0.193 respectively. Hardy-Weinberg equilibrium not observed in the herds 1, 2, 3 and 5. Shanno and Nei’s diversity indices showed that herds 4 and 1 are at lowest and highest gene diversity, respectively. Association between lactoferrin gene and productive and performance traits was analyzed by GLM procedure (SAS software). Results showed significant associations between LTF and fat percentage, conception rate and somatic cell score (SCS). However, association between genotypes and others such as adjusted 305-day milk, pregnancy length, lactation length and protein percentages was not significant (P 0.05). Key words: Lactoferrin gene, Polymorphism, performance traits, dairy cows.