In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm ) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010-2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.