This study was conducted to analyze the specific genes associated with sex-determination in semen of Korean Native cow. Male fertility is dependent upon the successful perpetuation of spermatogenesis that is a highly organized process of germ cell differentiation occurring within the seminiferous tubules in the testis. The highly organized spermatogenesis requires accurate, spatial and temporal regulation of gene expression governed by transcriptional, post-transcriptional and epigenetic processes. Recently, the farmers have been interesting in the male or female of calves in the their farm. In first, we analyzed the semen supplied from Hanwoo Improvement Center, NongHyup. The sperm motility in Hanwoo was decreased approximately 10 % in the 30 min after sex-determinant reagent. However, Holstein' sperm motility was decreased to 60-70% after 15 min and the motility was considerably decreased to 20-30 % after 30 min. Next, we analyzed the sperm specific expression genes both male- and female reagents treated-group. The real-time PCR results suggest that the selected genes (GIMP4, TMEFF1, RAC2, ABI2, RAC1, and CLUS) were highly expressed in the group treated with the male reagent compared to female reagent treated group and untreated-group. In the present study, although X or Y gene is play a key role in the sex-determination of mammalian, we suggest that the selected genes may be involved in the sex-determination.