Microcystins (MCs) are toxic heptapeptides found in cyanobacteria and share the common structure cyclo(-D-Ala1-L- X2-D-isoMeAsp3-L-Z4-Adda5-D-isoGlu6-Mdha7). The letters X and Z in the general formula above represent a wide range of L-amino acids that occupy positions 2 and 4, respectively. In general the variation in structural variants is due to the exchange of amino acids in position 7, 2, and 4. In the present work we report two homotyrosine (Hty)-containing microcystin variants, [D-Asp3,(E)-Dhb7]-MC-HtyY (1) and [D-Asp3,(E)-Dhb7]-MC-HtyHty (2), which were isolated from strain No80 of Planktothrix rubescens. Their structures were elucidated using amino acid analysis as well as 1D and 2D NMR techniques. The adenylation domains of McyABC involved in amino acid activation in positions 7, 2, and 4 of the microcystin molecule, respectively, were compared with corresponding genes of Planktothrix strain CYA126/8 producing [D-Asp3,Mdha7]-MC-RR and [D-Asp3,Mdha7]-MC-LR. While the adenylation domain comparison of McyAB between the two Planktothrix strains revealed considerable DNA recombination, the adenylation domain of McyC showed only a single amino acid substitution, which was correlated with the replacement of Arg by Hty in position 4 of the microcystin molecule.