ABSTRACT We have developed a procedure to test the efficiency and reliability of sequencing of Legionella pneumophila genes directly from respiratory samples and have compared the results with those derived from cultured isolates. We tried to obtain the nucleotide sequences of six protein-coding loci included in the sequence-based typing scheme for Legionella pneumophila and three intergenic regions from 132 samples corresponding to 106 patients positive for urine antigen. A seminested PCR approach was used to amplify and sequence these nine loci directly from respiratory samples. Nucleotide sequences were directly obtained for 23 Legionella isolates and also for 66 respiratory secretions from a total of 69 patients. The efficiency of sequencing from respiratory secretions was higher than that of sequencing after the isolation of the Legionella isolates. Moreover, the perfect match between the sequences obtained by both approaches when respiratory samples and cultured isolates from the same patient were available corroborates the suitability of the direct sequencing approach for the identification of Legionella species and molecular epidemiology studies with Legionella species.