Legionella longbeachae is almost as frequent a cause of legionellosis in Australia as Legionella pneumophila, but epidemiological investigation of possible environmental sources and clinical cases has been limited by the lack of a discriminatory subtyping method. The purpose of this study was to examine the genetic variability among Australian isolates of L. longbeachae serogroup 1. Pulsed-field gel electrophoresis (PFGE) of SfiI fragments revealed three distinct pulsotypes among 57 clinical and 11 environmental isolates and the ATCC control strains of L. longbeachae serogroups 1 and 2. Each pulsotype differed by four bands, corresponding to <65% similarity. A clonal subgroup within each pulsotype was characterized by >88% similarity. The largest major cluster was pulsotype A, which included 43 clinical isolates and 9 environmental isolates and was divided into five subgroups. Pulsotypes B and C comprised smaller numbers of clinical and environmental isolates, which could each be further divided into three subgroups. The ATCC type strain of L. longbeachae serogroup 1 was classified as pulsotype B, subtype B3, while the ATCC type strain of L. longbeachae serogroup 2 was identified as a different pulsotype, LL2. SfiI macrorestriction analysis followed by PFGE showed that the Australian L. longbeachae strains are not a single clonal population as previously reported.