Harmful cyanobacterial blooms, especially Microcystis blooms, occur worldwide and draw widespread attention. The dynamics of microcystin-producing Microcystis and competition between microcystin-producing Microcystis and non-microcystin-producing Microcystis are key to predicting and treating Microcystis blooms. Multiplex qPCR is a useful tool to assess such issues. In this study, we developed multiplex qPCR methods with newly-designed probes and primers for the microcystin-synthesis related genes mcyA and mcyE. We used seven toxic Microcystis strains and four non-toxic Microcystis strains to compare the differences in the ratios of toxic and non-toxic Microcystis in mixed cultures, which were calculated using abundances of the genes mcyA, mcyB, mcyD, mcyE and phycocyanin (PC). We also compared traditional cell counting and multiplex qPCR. Hierarchical clustering and principal component analysis indicated that mcyD was the most suitable mcy gene for quantification in laboratory experiments. mcyB abundances were always higher; we suggest that the amount of toxic Microcystis measured using mcyB might overestimate the actual percentages.