Currently, two distinct lineages of influenza B virus (IBV), B/Victoria and B/Yamagata lineage, have been co‐circulating in human beings. Assessment of the prevalent lineage is key for the recommendation of the seasonal influenza vaccine composition and the evaluation of its efficacy. In this study, a multiplex qRT‐PCR assay for the discrimination of the IBV lineages was designed based on the genetic differences of the hemagglutinin genes between B/Yamagata and B/Victoria lineages. The assay was highly specific and able to discriminate the lineages of IBV without any non‐specific reaction against other influenza A viruses. The detection limit of the assay was determined to be 10 genome‐equivalent copies and 2.8 × 10^‐2 50% tissue culture infectious doses (TCID₅₀) of live IBV per reaction. Moreover, our assay was able to discriminate the lineages of IBVs in clinical samples with 100% accuracy, when compared with pyrosequencing. Our results indicate that this assay may represent an update of the existing qRT‐PCR assays and will be of great use for the rapid and accurate diagnosis and surveillance of the circulating IBVs.