An important purpose in dairy cattle breeding is reaching to an efficient and economic benefit without increasing the herd size. Milk and other dairy products are the most important parts of human foods. Therefore, increasing production and improvement of milk quality are always the main purpose in milk production industry. There are several selection methods which could be used to approach genetic improvement and increase the production and quality of milk products. New methods of selection like using genetic polymorphism of genes affecting production traits such as genetic markers along with other common methods of selection can accelerate the genetic improvement process. Also, increasing selection accuracy and reducing bias rate in evaluation of populations effectively. However, there are a lot of genes reported affecting milk production and milk contents which can be used as genetic markers such as beta-lactoglobulin gene. Beta-lactoglobulin is a small globular protein of whey protein in ruminants' milk which is present in some non-ruminants' milk too. Previous studies have shown this protein is a polymorph in most of dairy cattle breeds which is due to simple replacement of a base pair in beta-lactoglobulin gene which could be simply identified by PCR-PFLP technique. The purposes of this study were to determine the frequency of polymorphism at this locus and its effect on production traits of Iranian Holstein cattle. Therefore, a total of 234 blood samples were collected from four different farms in Isfahan city and genomic DNA was extracted by modified Miller's method. PCR-RFLP technique was used to determine the genotypes. The segment including the mutation resulting in polymorphism was amplified by specific primers. Then 262 bp product was digested by Hae III restriction enzyme. Digested product was loaded on 16% non denaturing polyacrylamide gel. Presence of segments with 109 and 153 bp referred to A allele and segments with 109, 79 and 74 bp referred to B allele. The results of allelic and genotype frequencies showed that B allele (0.525) and AB genotype (0.41) were most frequent ones. To study the effects of genotype, SAS software (Proc GLM) was employed. In the linear model of analysis, the effects of herd and genotype were considered as the fixed effects and days in lactation, dry period and open days were considered as the covariables. Among milk production and milk contents traits, reproduction and birth weight, genotypes were only significantly effective on birth weight (P 0.05) and crude milk production (P 0/10). AA genotype had the highest relationship with higher crude milk production as well as higher birth weight which might be considerd in selection index for suitable genotype selection of these traits.