Introduction. Pestiviruses are the cause of reproductive problems, diseases of the gastrointestinal and respiratory tracts of animals. Three species are important for cattle: Pestivirus A, B, and H. Fast and reliable methods of differentiation of these pathogens are currently needed.Aims and objectives of the study: the development of multiplex real time PCR for the simultaneous detection and differentiation of three viruses.Material and methods. The nucleotide sequences of the conserved regions of the 5´-UTR genes of pestiviruses A, B, and H served as a target.Results. The reaction showed high specificity, sensitivity, reproducibility and was able to detect virus RNA at a concentration of not less than 0.6-1.2 lg TCID50/cm3. Cross-reactions with other pestiviruses were not observed. Real time PCR confirmed the results obtained previously in RT-PCR with gel electrophoresis detection. In a parallel study of 1823 biological samples, the results of the two reactions were completely consistent. Pestivirus spp. was detected in 76 samples, Pestivirus A was present in 73 samples, Pestivirus B - in 3 samples, and Pestivirus H was not detected.Discussion. A two-step real time PCR was developed for the simultaneous detection and differentiation of three pestiviruses. Modified pan primers of S. Vilcek et al. were used for the first reaction, and primers and probes of our own design were used for virus typing, which resulted in high reaction efficiency.Conclusion. On the big dairy farms for livestock maintenance, there are favorable conditions for the circulation of pathogenic viruses. In this situation, rapid diagnostic methods are needed to quickly identify of several viruses. Real-time triplex analysis can be recommended as the rapid method for mass epidemiological studies, as well as for screening fetal calf serum used for virus cultivation in medicine and veterinary practice.