The H1N1 subtype influenza viruses (H1N1) have been causing persistent epidemics in human, swine and poultry populations since 1918. This subtype has evolved into four relatively stable genetic lineages, including classical swine influenza virus lineage, seasonal human influenza virus lineage, avian influenza virus lineage and Eurasian avian-like swine influenza virus lineage. In this study, four pairs of primers, based on the relatively conserved HA nucleotide regions of each H1N1 genetic lineage, were designed to establish a SYBR Green-based real-time quantitative RT-PCR (qPCR) assay to differentiate between the H1N1 genetic lineages. The results of qPCR assay showed that the lineage-specific primers designed for each H1N1 lineage were intra-lineage-specific, without mismatch of inter-lineage or inter-subtype and there appeared specific amplification curves when the concentrations of H1N1 plasmids were greater than or equal to 1.0 × 10 copies/reaction. Thus, this qPCR assay can specifically differentiate between the four lineages of H1N1 with a good specificity and sensitivity, which would assist in recognizing the infection and epidemic status of different H1N1 genetic lineages.