The amount of information that is available on 16S rRNA sequences for prokaryotes thanks to high-throughput sequencing could allow a better understanding of diversity. Nevertheless, the application of predetermined threshold in genetic distances to identify units of diversity (Operative Taxonomic Units, OTUs) may provide biased results. Here we tests for the existence of a barcoding gap in several groups of Cyanobacteria, defining units of diversity according to clear differences between within-species and among-species genetic distances in 16S rRNA. The application of a tool developed for animal DNA taxonomy, the Automatic Barcode Gap Detector (ABGD), revealed that a barcoding gap could actually be found in almost half of the datasets that we tested. The identification of units of diversity through this method provided results that were not compatible with those obtained with the identification of OTUs with threshold of similarity in genetic distances of 97% or 99%. The main message of our results is a call for caution in the estimate of diversity from 16S sequences only, given that different subjective choices in the method to delimit units could provide different results.