Abstract: A rapid, highly reproducible and sensitive technique has been successfully developed for sexing the cow embryos, by method of polymerase chain reaction (PCR) against the amelogenin (bAML) genes located on both X- and Y-chromosomes of the Holstein dairy cattle. Results from DNA sequence analysis showed that there was only 45% homology between the intron 5 of AMLX and AMLY genes. Based on these sequences a pair of sex-specific primers, pbAML5XY(+) and pbAML5XY (-),were designed allowing to amplify a single fragment of 476-bp from the female cattle and two fragments of 476-bp and 341-bp from the male ones, respectively. The most important feature is that the precise sensitivity of sex-determination was confirmed to be reached as minimum template as trace amount of genomic DNA content in either a single lymphocyte or a single blastomere isolated from cow embryo at day-6 to day-7.