<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of <jats:italic>mcyB (mcyB1)</jats:italic> and <jats:italic>mcyC</jats:italic> in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus <jats:italic>Microcystis</jats:italic>. </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Here we undertook a phylogenetic study to investigate the order and timing of recombination between the <jats:italic>mcyB1</jats:italic> and <jats:italic>mcyC</jats:italic> genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the <jats:italic>mcyB1</jats:italic> and <jats:italic>mcyC</jats:italic> adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between <jats:italic>mcyB1</jats:italic> and <jats:italic>mcyC</jats:italic> in strains of the genera <jats:italic>Anabaena</jats:italic>, <jats:italic>Microcystis</jats:italic>, and <jats:italic>Hapalosiphon</jats:italic>. We also find clear evidence for independent adenylation domain conversion of <jats:italic>mcyB1</jats:italic> by unrelated peptide synthetase modules in strains of the genera <jats:italic>Nostoc</jats:italic> and <jats:italic>Microcystis</jats:italic>. The recombination events replace only the adenylation domain in each case and the condensation domains of <jats:italic>mcyB1</jats:italic> and <jats:italic>mcyC</jats:italic> are not transferred together with the adenylation domain. Our findings demonstrate that the <jats:italic>mcyB1</jats:italic> and <jats:italic>mcyC</jats:italic> adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. </jats:sec>