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Accurately identifying the species present in an ecosystem is vital to lake managers and successful bioassessment programs. This is particularly important when monitoring cyanobacteria, as numerous taxa produce toxins and can have major negative impacts on aquatic ecosystems. Increasingly, DNA-based techniques such as metabarcoding are being used for measuring aquatic biodiversity, as they could accelerate processing time, decrease costs and reduce some of the biases associated with traditional light microscopy. Despite the continuing use of traditional microscopy and the growing use of DNA metabarcoding to identify cyanobacteria assemblages, methodological comparisons between the two approaches have rarely been reported from a wide suite of lake types. Here, we compare planktonic cyanobacteria assemblages generated by inverted light microscopy and DNA metabarcoding from a 379-lake dataset spanning a longitudinal and trophic gradient. We found moderate levels of congruence between methods at the broadest taxonomic levels (i.e., Order, RV=0.40, p0.0001). This comparison revealed distinct cyanobacteria communities from lakes of different trophic states, with Microcystis, Aphanizomenon and Dolichospermum dominating with both methods in eutrophic and hypereutrophic sites. This finding supports the use of either method when monitoring eutrophication in lake surface waters. The biggest difference between the two methods was the detection of picocyanobacteria, which are typically underestimated by light microscopy. This reveals that the communities generated by each method currently are complementary as opposed to identical and promotes a combined-method strategy when monitoring a range of trophic systems. For example, microscopy can provide measures of cyanobacteria biomass, which are critical data in managing lakes. Going forward, we believe that molecular genetic methods will be increasingly adopted as reference databases are routinely updated with more representative sequences and will improve as cyanobacteria taxonomy is resolved with the increase in available genetic information.