Influenza viruses type A and type B are a leading cause of annual epidemics in human populations. Since the 1970s, influenza B viruses have diverged into two antigenically distinct virus lineages called the Yamagata and Victoria lineages. We describe the validation and implementation of a one-step real-time RT-PCR (rRT-PCR) assay that can differentiate between the two genetic lineages of type B.Validation of rRT-PCR method was carried out using quantified positive control and reference influenza viruses with specific minor groove binder (MGB) probes. The assay was applied on 102 clinical specimens detected positive for influenza type B.Detection limit was found to be as low as 7.95 RNA copies per reaction. The interassay variability and intra-assay variability were found to be low, and comparable for Yamagata and Victoria lineages. No cross-reactivity with the tested subtypes of influenza type A, known to cause human infections, was noticed. Differentiation of influenza B lineages by rRT-PCR was successfully achieved on all of the known positive type B samples. From the total number of clinical specimens tested, 85 samples belonged to B/Yamagata and 17 samples to B/Victoria lineage.Differentiation of genetic lineage B influenza virus circulating in Romania in the next seasons by one-step real-time RT-PCR method will supplement the classical test, haemagglutination inhibition (HI), which requires growing of the virus. This method can be advantageous for a balanced selection of samples, in case of lineages co-circulation, for genetic and antigenic characterization.