Microcystin (MC)-producing Microcystis strains from environmental samples were assessed by the simultaneous amplification of up to five DNA sequences, corresponding to specific regions of six mcy genes (mcyA, mcyB, mcyC, mcyD, mcyE and mcyG), codifying for key motifs of the non-ribosomal peptide synthetase and polyketide synthase of the microcystin synthetase complex. Six primer pairs with the same melting temperature, one of them of new design, were used. A crucial point for the good performance of the new multiplex PCR test was the concentration of each primer pair. In the test, cell suspensions from laboratory cultures, field colonies and blooms were directly used as DNA source. The results of the multiplex PCR were consistent with the toxinogenic character of the samples, as checked by high performance liquid chromatography and/or matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. As a whole, the newly developed test could be used for a reliable, rapid and low-cost screening of potential MC-producing Microcystis in field samples, even scattered colonies.