Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive means to detect these toxins, although cross-reactivity characteristics of different antibodies are variable, and most antibodies have been produced against MC-LR. Here, we have produced the first polyclonal antibodies against the commonly occurring variant, MC-RR, and compared them with MC-LR antibodies for the analysis of purified MCs and cyanobacterial environmental samples. Both antisera cross-reacted with all MCs tested, and with the related cyanobacterial hepatotoxin nodularin-R, but not with non-toxic cyanobacterial peptides. In general, better cross-reactivity characteristics were observed with the MC-RR antisera and limits of quantification were lower for most variants, with all MCs tested and nodularin-R having limits of quantification of 0.31 nM or below. The antisera had different affinities to mixtures containing pooled MC-LR and MC-RR, with MC-LR antisera underestimating total MC concentration when MC-RR represented over 70% of the total MC pool. Both antisera correlated well with HPLC-UV data when incorporated into ELISAs to screen previously characterised environmental samples from Åland, Finland. MC-RR antisera are useful for screening samples containing multiple MCs, and particularly for samples primarily containing MC-RR variants.