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We reconstructed cyanobacterial community structure and phylogeny using DNA that was isolated from layers of stratified sediments spanning 200 years of lake history in the perialpine lakes Greifensee and Lake Zurich (Switzerland). Community analysis based on amplification and sequencing of a 400-nucleotide (nt)-long 16S rRNA fragment specific to Cyanobacteria revealed operational taxonomic units (OTUs) capturing the whole phylum, including representatives of a newly characterized clade termed Melainabacteria , which shares common ancestry with Cyanobacteria and has not been previously described in lakes. The reconstruction of cyanobacterial richness and phylogenetic structure was validated using a data set consisting of 40 years of pelagic microscopic counts from each lake. We identified the OTUs assigned to common taxa known to be present in Greifensee and Lake Zurich and found a strong and significant relationship (adjusted R 2 = 0.89; P < 0.001) between pelagic species richness in water and OTU richness in the sediments. The water-sediment richness relationship varied between cyanobacterial orders, indicating that the richness of Chroococcales and Synechococcales may be underestimated by microscopy. PCR detection of the microcystin synthetase gene mcyA confirmed the presence of potentially toxic cyanobacterial taxa over recent years in Greifensee and throughout the last century in Lake Zurich. The approach presented in this study demonstrates that it is possible to reconstruct past pelagic cyanobacterial communities in lakes where the integrity of the sedimentary archive is well preserved and to explore changes in phylogenetic and functional diversity over decade-to-century timescales. IMPORTANCE Cyanobacterial blooms can produce toxins that affect water quality, especially under eutrophic conditions, which are a consequence of human-induced climate warming and increased nutrient availability. Lakes worldwide have suffered from regular cyanobacterial blooms over the last century. The lack of long-term data limits our understanding of how these blooms form. We successfully reconstructed the past diversity of whole cyanobacterial communities over two hundred years by sequencing genes preserved in the sediments of two perialpine lakes in Switzerland. We identified changes in diversity over time and validated our results using existing data collected in the same two lakes over the past 40 years. This work shows the potential of our approach for addressing important ecological questions about the effects of a changing environment on lake ecology.