16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains, with 98.65% similarity currently recognized as the cutoff for delineating species. The comparison of the 16S rRNA gene sequence of an isolate against sequences of type strains of all prokaryotic species provides an accurate and convenient way to routinely classify and identify prokaryotes. Here, we present the theoretical background to detecting phylogenetic neighbours and for calculating pairwise similarities between 16S rRNA gene sequences, using a process named the “EzTaxon search”. In addition, a workflow procedure is introduced that allows the analyses necessary for the identification of prokaryote strains, starting from raw Sanger sequencing data to the generation of phylogenetic trees, using the EzTaxon server (http://www.ezbiocloud.net/eztaxon) and EzEditor software. The application of these procedures will ensure best practice in manual editing of 16S rRNA sequence data and their use for taxonomic purposes.