Blood is the standard source for DNA analysis, but requires venipuncture of cows by veterinarian and tedious and costly DNA extraction. A procedure was developed for sampling of vaginal cells from cows, establishing a cell lysis protocol using robotics, and applying fluorescent analysis of genetic markers. Two insemination technicians collected vaginal cells from 254 elite Israeli Holstein cows located in 152 herds using commercial Catch-All sample collection brushes. Cells were lysed in a 400-microliters solution, and 5 microliters was used as template for polymerase chain reaction (PCR). Sensitivity of the PCR was enhanced using only 1 microliter of lysed cells. Eight markers of the International Society of Animal Genetics paternity panel were amplified in four separate PCR. ILSTS039, a marker for a quantitative trait loci on BTA14, was amplified in a separate reaction. Genotypes from one to nine genetic markers were obtained for 253 out of 254 samples, and 244 cows had genotypes for at least three markers (96%). Cows that did not inherit either paternal allele for at least two loci were considered not to be daughters of the sire listed. Fifteen cows met this criterion, for a paternity misidentification rate of 6.25%. The frequency of allele 225 of ILSTS039, which was associated with increased milk fat content, was 11.1% in the bull-dam population, similar to the 12% found in the cow population in Israel. The use of vaginal cells for genetic analysis is accurate, as demonstrated by replicated analysis and by comparison to individual and population analysis based on DNA derived from blood.