The genus Legionella is represented by 48 species and Legionella pneumophila includes 15 serogroups. In this work, we have studied the intergenic 16S-23S spacer region (ITS) in L. pneumophila to determine the feasability of using amplification polymorphisms in this region, to establish intraspecies differences and to discriminate Legionella species. The amplification of this region, using 16S14F and 23S0R primers, and the analysis of amplicons by the analysis of fragments technique showed that all the L. pneumophila serogroups studied presented the same electrophoretic pattern. Moreover, the analysis of different Legionella species showed that the amplification polymorphisms obtained were species-specific. In order to study the sequence variability of this region, the existence in L. pneumophila of three ribosomal operons was determined by pulsed field gel eletrophoresis (PFGE). Two of the 16S-23S rRNA ITS presented a tRNA Ala and the third one a tRNA Ile. Nevertheless, the variability expected in this region of the operon was not found and the rest of the ITS contained only punctual mutations.