This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability ($84{pm}1.15%$, $p$<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability ($79{pm}3%$, $p$<0.05) than those of the X and Y sperm ($44.7{pm}1.67$ and $41.7{pm}1.2%$) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities ($24.3{pm}1.2$ and $25.7{pm}0.9%$, $p$<0.05) compared to that of the sorted Y sperm ($13.7{pm}1.2%$) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher ($55.0{pm}1.7$ and $45.0{pm}1.5%$) than those of the sorted Y-sperm ($32.3{pm}0.9%$, $p$<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.