Despite the crucial role of cyanobacteria in various ecosystems, little is known about their evolutionary histories, especially microevolutionary dynamics, because of the lack of knowledge regarding their mutation rates. Here we directly estimated cyanobacterial mutation rates based on ancient DNA analyses of ice core samples collected from Kyrgyz Republic that dates back to ~12,500 cal years before present. We successfully sequenced the 16S rRNA and 16S-23S internal transcribed spacer (ITS) region. Two cyanobacterial operational taxonomic units (OTUs) were detected from the ancient ice core samples, and these OTUs are shared with those from the modern glacier surface. The mutation rate of ITS region was estimated by comparing ancient and modern populations, and were at the magnitude of 10-7substitutions/sites/year. By using a model selection framework, we also demonstrated that the ancient sequences from the ice sample were not contaminated from modern samples. Bayesian demographic analysis based on coalescent theory revealed that cyanobacterial population sizes increased over Asia regions during the Holocene. Thus, our results enhance our understanding of the enigmatic timescale of cyanobacterial microevolution, which has the potential to elucidate the environmental responses of cyanobacteria to the drastic climatic change events of the Quaternary.