Analysis of PCR-amplified transfer DNA (tDNA) intergenic spacers was evaluated as a rapid method for identification to the species level of 18 species of Legionella known as human pathogens. Type strains (n = 19), reference strains (n = 16), environmental strains (n = 31), and clinical strains (n = 32) were tested. PCR products using outwardly directed tDNA consensus primers were separated on polyacrylamide gels and analyzed with automated laser fluorescence. Test results were obtained in 8 h starting with 72-h-old bacterial growth on solid medium. Species-specific patterns were obtained for all 18 Legionella species tested: Legionella anisa, L. bozemanii serogroups 1 and 2, L. cincinnatiensis, L. dumoffii, L. feeleii serogroups 1 and 2, L. gormanii, L. hackeliae serogroups 1 and 2, L. jordanis, L. lansingensis, L. longbeachae serogroups 1 and 2, L. lytica, L. maceachernii, L. micdadei, L. oakridgensis, L. parisiensis, L. pneumophila serogroups 1 to 14, L. sainthelensi serogroup 2, L. tucsonensis, and L. wadsworthii. Computer-assisted matching of tDNA-intergenic length polymorphism (ILP) patterns identified all 63 environmental and clinical strains to the species level and to serogroup for some strains. tDNA-ILP analysis is proposed as a routinely applicable method which allows rapid identification of environmental and clinical isolates of Legionella spp. associated with legionellosis.