Sex preselection in cats can have applications for both breeding purposes and as an experimental model for endangered felids. The present study examined the ability to produce cat embryos from in vitro fertilization (IVF) of in vitro matured (IVM) cat oocytes with flow cytometrically sorted spermatozoa and to verify the sex of the embryos obtained from sexed spermatozoa by PCR. In the first experiment, a total of 224 oocytes were fertilized with spermatozoa from six ejaculates sorted without sex separation. The sorting process did not influence the cleavage rate (sorted 44.0% versus unsorted 46.1%), day 6 morula-blastocyst rate (sorted 26.6% versus unsorted 29.6%) and day 7 blastocyst rate (sorted 16.5% versus unsorted 16.5%). In the second experiment, a total of 84 IVM oocytes were fertilized with sorted X- and Y-chromosome bearing spermatozoa from four ejaculates in order to obtain embryos of preselected sex. Embryonic sex determination by PCR revealed that 21 out of 24 embryos reaching morula/blastocyst stage (87.5%) were of the desired sex. In particular 12 out of 14 embryos (85.7%) derived from X-bearing spermatozoa were female and 9 embryos out of 10 (90%) derived from Y-bearing spermatozoa were male. Our results show, for the first time, that X- and Y-chromosome bearing spermatozoa sorted by high-speed flow cytometry can be successfully used in an IVM-IVF system to obtain cat embryos of a predetermined sex.