Background We investigated the diversity of the primary sequences of the 16S rRNA genes among 46 commensal Neisseria strains and evaluated the use of this approach as a molecular typing tool in comparison with PFGE analysis. Methods Identification to the genus was done using conventional methods and API NH (bio‐Mérieux®). Identification to species level was based on 16S rRNA gene sequencing. PFGE analysis was done using SpeI. Results Fourteen, two, three and fourteen 16S rRNA sequence types were found among twenty Neisseria flavescens, two Neisseria sicca, five Neisseria macacae and nineteen Neisseria mucosa clinical isolates. Forty‐three different PFGE patterns were found among the tested strains. Conclusion We demonstrated a high diversity among 16S rRNA genes which was reflected by PFGE analysis.