In this work, we describe a SYBR-Green one-step RT-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 131pb amplicon, obtained from a conserved region of hemagglutinin (HA) 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 profile. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test p<0.0001, CI 95%) and the Tm is not affected by viral load (Spearman Correlation test r = 0.287, p= 2.245x10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.