This experiment was conducted to evaluate the assessment of alpha-1-anti trypsin gene polymorphism and its association with production traits in Holstein dairy cows. Alpha-1-antitrypsin (AAT), a strong protease inhibitor, also known as ?1-protease inhibitor, belongs to the superfamily of serpins, or serine proteinase inhibitors that include amongst others C1 esterase, antithrombin and ?1-antichymotrypsin. AAT is a glycoprotein which forms a SDS stable complex with elastase. The molecular mass of AAT is about 52 kDa, and carbohydrates account for 15% of its mass. The bovine AAT gene consists of five exons, spanning about 9 kb of genomic DNA and encoding a 416-AA protein. Five at positions 164, 269, 284, 407, and 989 of the bovine proteinase inhibitor gene were identified in different cattle populations. The major role of AAT is to protect tissue against proteolytic digestion by neutrophil elastase. The nucleotide substitutions in this positions leads to create or loss restriction enzyme recognition site and using this we can identify different alleles of this gene. Blood samples obtained from a random sampling of dairy Holstein cows maintained in five farms located in Isfahan province (n=408) to investigate polymorphism of AAT gene. Number of samples varied from 65 to 96 among herds. DNA was extracted using salting out procedure. The quality and quantity of DNA determined by electrophoresis. The 448 bp fragment of this gene amplified using specific primers by PCR. PCR product was detected by electrophoresis on 1% TAE agarose gel. Amplified fragment digested using SphI restriction enzyme for 16 hours at 37 ? c. The genotype frequency of AA, AB, and BB were 0.36, 0.55, and 0.09, respectively. Allele A of the AAT gene occurred at a higher frequency than allele B. The frequencies of alleles A and B alleles were 0.63 and 0.37 respectively. The A allele in Herd 5 was more frequent than other herds but the A allele in Herd 2 was lowest frequency. In general, five herds were not in Hardy-Weinberg equilibrium. The Shannon and Nei’s index showed herd number 5 has lowest and herd number 2 has highest gene diversity among the studied herds. In this study the association between AAT genotypes and productive and performance traits was analyzed by GLM procedure of SAS software. The results showed significant associations between the genotypes, with fat and protein percentages. No significant association was found between genotypes and adjusted 305 day milk, lactation period and SCS (P 0.05). These results suggest that AAT is a candidate gene that influences milk production traits and it could be implemented in breeding programmes to improve the production performance of Holstein cattle. Key words: Alpha-1-antitrypsin gene, polymorphism, Holstein cow, genotype