Sex determination of anonymous samples is a requirement before analysis of DNA variation on X or Y chromosomes. Based on this, we designed a method for screening samples on different DNA capillary sequencing instruments with a sensitivity that is able to quantify sex chromosome abnormalities. The two different amelogenin alleles sited on the X and Y chromosomes were polymerase chain reaction amplified with the same set of primers and separated by denaturant capillary electrophoresis (DCE). Sex chromosome ratios could be reproducibly determined with a relative standard deviation of 8.7%, which is sufficient to distinguish a normal XY karyotype from an XYY karyotype associated with Klinefelter syndrome. Reconstruction experiments demonstrated sensitivity down to a simulated Y:X allelic ratio of 1:127 in all three instruments, enabling the prediction of sex chromosomal aneuploidies. When tested on anonymous pooled and single samples, DCE gave a good prediction of the male to female ratio in pools of 1000 blood donors. In conclusion, DCE is a simple and robust method for sex determination that can be readily performed on commercially available CE systems.