Abstract: The polymorphisms in intron 11，19，25 and 3′UTR of STAT1 gene were detected in 199 Chinese Holstein cattles with PCR-SSCP and association between the polymorphisms and milk production traits of three birth orders were analyzed. There were three polymorphism sites in Chinese Holstein cattles, which were in intron11 and 3′UTR. We found that it was G/A (28739) and G/C (28873) in intron 11, and C/T (141930) in 3′UTR. The association between different genotypes of three sites and 305 days milk yield, fat percent (F%), and protein percent (P%) of three birth orders was analyzed by least square analysis. The results showed that in intron 11，there were no significant differences among three genotypes on 305 days milk yield, and fat percent(F%)of all birth orders(P>0.05), but the tendency of BB>AB>AA was showed. Cows with genotype BB had significantly higher protein percent (P%) in the first and second birth orders (P<0.05) than those with AB and AA. And in 3′UTR, there were no significant differences among three genotypes on fat percent (F%) (P>0.05), while the tendency of CC>CT>TT was showed either. And cows with genotype CC or CT had significantly higher 305 days milk yield in the first and second birth orders (P<0.05); and cows with genotype CC had significantly higher protein percent (P%) than those with genotype TT in the first and third birth orders(P<0.05).