Despite the dominance of cyanobacteria in polar freshwater aquatic ecosystems, little is known about their past biodiversity and response to climate and environmental changes. We explored the use of light microscopy of microfossils, high performance liquid chromatography of the fossil pigment composition and denaturing gradient gel electrophoresis of fossil 16S rRNA genes to study past and present-day differences in cyanobacterial community structure in response to climate changes in two adjacent maritime Antarctic lakes with contrasting depths (4 and 26 m) and light climates. Light microscopy was of limited use because of degradation of cell structures. Fossil cyanobacterial pigment concentrations were below the detection limits of our method in several sediment samples in the deep lake, but abundant and diverse in the sediment core from the shallow pond, probably as a consequence of increased light availability and/or a more diverse and abundant benthic cyanobacterial flora. Total carotenoid and chlorophyll concentrations were highest in both lakes between ca. 2,950 and 1,800 cal yr BP, which coincides with the late Holocene climate optimum recognised elsewhere in maritime Antarctica. Cyanobacterial molecular diversity was higher in the top few centimeters of the sediments in both lakes. In deeper sediments, the taxonomic turnover of cyanobacteria appeared to be relatively small in response to past climate anomalies in both lakes, underscoring the broad tolerance of cyanobacteria to environmental variability. This, however, may in part be explained by the low taxonomic resolution obtained with the relatively conserved 16S rRNA gene and/or the preferential preservation of particular taxa. Our results highlight the potential of fossil DNA in lake sediments to study colonization and succession dynamics of lacustrine cyanobacteria and warrant further investigation of the factors that affect preservation of cyanobacterial DNA.