The freemartinism is the most frequent form of intersexuality found in cattle, and females of heterosexual twins become sterile. With increase of twinning rates due to transfer of multiple embryos derived from in vitro fertilization, it is of great economic value to establish early diagnosis of freemartins to remove infertile individuals from breeding stock. In the present study polymerase chain reaction (PCR) of two different Y-chromosome specific segments (BRY.l and AMX/Y) was performed to identify freemartins from twins and less common single born freemartins in Korean Native Cattle (KNC). Two male-specific sequences were amplified in all heterosexual twins tested (n=5). In addition, Y-specific PCR products were detectable in one of the single born females (n=4) with visible genital abnormalities. These results suggest that the sensitivity of PCR-based assay may be sufficient to detect freemartinism in single born females as well as female partners of heterosexual twins in KNC.