Widely used bovine sexing primers were compared in terms of suitability in determining the sex of bovine embryos. Under optimized multiplex PCR conditions, the ConBV/ConEY couple primers did not show accurate results when combined together in multiplex PCR, but worked well when the couple primers were used separately. The S4BF/S4BR primers showed accurate results; however, some unexpected bands were detected. When the BY/BSP couple primers were used to determine one-cell, two-cell, four-cell and eight-cell stage embryos of known sexed SCNT-derived embryos, the results showed 100% accuracy. The BY/BSP couple primers were also able to identify the sex of one-cell and two-cell IVF-derived embryos.