Abstract: In order to identify the genetic markers associated with milk production and mastitis resistance, and provide theoretical basis for molecular breeding, 383 Chinese Holstein cows (all were the 2nd parity) were chosen as study animals. Direct sequencing and time of flight mass spectrometry were used to find polymorphisms in 5' and 3'-flanking region of TBX21 gene. Then association of the two polymorphic sites with the 4 milk production traits including milk yield, fat percentage, protein percentage and lactose percentage and SCC (somatic cell count) was analyzed. The results showed that two polymorphic sites were detected in 5' and 3'-flanking region of TBX21 gene. There were one A→G mutation at 1091 bp and one A→C mutation at 654 bp in 3'-flanking region. Both of them were in Hardy-Weinberg equilibrium. Association analysis showed that the 4 milk production traits and somatic cell count of rs207725545 loci AA genotype were higher than those of CC genotype (P>0.05). For rs110420592, milk yield and lactose percentage of GG genotype were higher than those of AA genotype (P>0.05), fat percentage and protein percentage of AA genotype were higher than those of GG genotype (P>0.05), and somatic cell count of GA genotype were higher than that of GG and AA genotype (P>0.05). The two loci of rs110420592 and rs207725545 in TBX21 gene had no significant association with the milk production traits and somatic cell count. Analysis of genetic effects on other polymorphic sites of TBX21 gene was needed in the future.