In the human, mosaicism may occur before implantation; but, to determine when it first occurs, it is necessary to study the chromosomal complement of all blastomeres. Full karyotypes of blastomeres from 2- to 8-cell human embryos by conventional karyotyping of metaphase spreads are difficult to obtain. The aim of this study was to assess the stage at which mosaicism occurred in preimplantation human embryos through use of fluorescence in situ hybridization (FISH) with multiple probes. All or most blastomeres from 2- to 12-cell human embryos were analyzed by FISH using probes for gonosomes and chromosome 18. FISH was performed on blastomeres from 117 morphologically normal monospermic embryos that were not transferred after preimplantation diagnosis because of their risk of carrying X-linked disease; 20 (17.1%) of these embryos were mosaic. Another group of 163 arrested or morphologically abnormal monospermic embryos were also analyzed by FISH; 47 (28.8%) of these embryos were mosaic. In addition, 37 dispermic embryos were analyzed, and 28 (75.7%) of these were found to be mosaic. Mosaicism first occurred at the second cleavage division when the monospermic embryo was mostly diploid and at the first cleavage division when the embryo was mostly haploid, polyploid, or dispermic.