The use of molecular methods to investigate the community structure and diversity of microalgae has largely replaced the previous morphological methods that were routinely carried out by microscopy. Different DNA polymerases can lead to bias in PCR amplification and affect the downstream community and diversity analysis. In this study, two clone libraries were constructed with two different DNA polymerases, Q5 high-fidelity DNA polymerase and exTaq polymerase, to compare the differences in their capability to accurately reflect the cyanobacterial community structure and diversity in a subtropical deep freshwater reservoir, Dongzhen reservoir. The results indicated that the two cyanobacterial clone libraries constructed by using Q5 high-fidelity DNA polymerase and exTaq DNA polymerase did not show significant differences, although a slightly higher community diversity was revealed by using Q5 high-fidelity DNA polymerase. It is noteworthy that so far Q5 high-fidelity DNA polymerase was the first time to be employed in the genetic analysis of cyanobacterial community. And it is for the first time that the cyanobacterial community structure in Dongzhen reservoir was analyzed using molecular methods. Phylogenetic analysis revealed that most of the clones belonged to Cyanophyta and chloroplast, among which Cyanobium sp. Suigetsu-CR5 made up the largest fraction of cyanobacteria in winter.