In this study we show that the hipervariable regions V5-V7 of the 16S rRNA gene is the most appropriated to study the diversity and structure of picocyanobacterial communities using Illumina MiSeq. Due to its variability allows the reconstruction of phylogenetic trees with comparable topologies to those generated by using the complete ribosomal gene sequence. We successfully designed a new set of primers flanking these regions whose specificity for picocyanobacterial genera was tested in silico and validated in several aquatic communities. The present work represents a step forward in the understanding of the diversity and ecology of picocyanobacteria in aquatic systems.