In search of a primer set that could be used to study Planctomycetes dynamics in lakes and especially via fingerprinting methods, e.g. denaturing gradient gel electrophoresis (DGGE), three existing specific primer sets, developed for marine and soil systems, have been tested on water samples from four freshwater ecosystems. The first primer set (PLA46F/PLA886R) allowed PCR amplification of Planctomycetes sequences in only one of the four ecosystems, whereas the second primer set (PLA40F/P518R) amplified Planctomycetes sequences in all the studied ecosystems but with a low specificity, since sequences belonging to Verrucomicrobiales and Chlamydiales clades were also amplified. Finally, the third primer set (PLA352F/PLA920R) allowed amplification of Planctomycetes sequences in the four ecosystems with a very high specificity. It amplified all known Planctomycetes genera and yielded the highest Operational Taxonomic Unit (OTU) richness and diversity estimates. In silico analyses supported these results. Further experiments comparing PLA352F/PLA920R to PLA46F/P1390R (a primer set generating a longer PCR fragment, also used to study Planctomycetes) yielded very similar results. Our findings suggest that the primer set PLA352F/PLA920R provides good estimates of Planctomycetes richness and diversity compared with other, and can thus be used to study Planctomycetes dynamics in lentic freshwater ecosystems.