Harmful cyanobacterial blooms pose a risk to human health worldwide. To enhance understanding on the bloom-forming mechanism, the spatiotemporal changes in cyanobacterial diversity and composition in two eutrophic lakes (Erhai Lake and Lushui Reservoir) of China were investigated from 2010 to 2011 by high-throughput sequencing of environmental DNA. For each sample, 118 to 260 cpcBA-IGS operational taxonomic units (OTUs) were obtained. Fifty-two abundant OTUs were identified, which made up 95.2% of the total sequences and were clustered into nine cyanobacterial groups. Although the cyanobacterial communities of both lakes were mainly dominated by Microcystis, Erhai Lake had a higher cyanobacterial diversity. The abundance of mixed Nostocales species was lower than that of Microcystis, whereas Phormidium and Synechococcus were opportunistically dominant. The correlation between the occurrence frequency and relative abundance of OTUs was poorly fitted by the Sloan neutral model. Deterministic processes such as phosphorus availability were shown to have significant effects on the cyanobacterial community structure in Erhai Lake. In summary, the Microcystis-dominated cyanobacterial community was mainly affected by the deterministic process. Opportunistically dominant species have the potential to replace Microcystis and form blooms in eutrophic lakes, indicating the necessity to monitor these species for drinking water safety.