Abstract: Polymorphism of kappa casein protein (6-casein) in total 54 Holstein cattle was investigated using Horizontal Starch-urea Gel Electrophoresis Method to verify its effect on milk production traits and influence of other factors such as parity and season of calving; aiming at utilizing it as a genetic aid in selection to improve the milk production traits of this breed. The allelice frequencies of A and B were found to be 0.352 and 0.648, respectively. Genotype frequencies were in accordance with the Hardy-Weinberg equilibrium. General Linear Model (GLM) was used to analyze differences between genotypes. The results indicated that 6-casein genotypes significantly (p<0.05) affected 305 days milk yield, 305 days fat yield and fat percentage. But it had no effect on actual milk yield, actual fat yield and lactation length. Parity number had significant effect on milk production traits. Season of calving had significant effect 305 days milk yield, actual fat yield, 305 days fat yield and fat percentage but it had no effect on actual milk yield and lactation length. This study indicated that the 6-casein genetic variants may be used as a genetic aid through increasing the frequency of desired genotypes to improve the milk production traits of this breed.