The potential effect of in-premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free-living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington-2 increased in growth at 30, 32 and 37°C while strains Philadelphia-1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington-2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia-1 in the presence of N. fowleri. Yet, strain Bloomington-2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human-pathogenic legionellae and point to future research required to better understand Legionella ecology. No claim to US Government works. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.