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To characterize Francisella isolated from two natural warm springs in Utah and compare them to a strain isolated from a patient with probable exposure to one of the springs in 2001.A total of 39 presumptive Francisella isolates were obtained from two springs, Wasatch Hot Spring and Hobo Warm Spring, just north of Salt Lake City, Utah. All isolates were characterized by a combination of biochemical and molecular analyses, including novel PCR/electrospray ionization-mass spectrometry (ESI-MS) typing assays. Thirty-one were identified as F. philomiragia, while the remaining eight were identified as F. tularensis ssp. novicida. Phylogenetic analysis of the 16S rRNA sequences revealed 27 isolates, which clustered with F. philomiragia, albeit into two distinct clades. The remaining isolates clustered along with other F. tularensis strains including the Utah clinical isolate. Testing with the PCR/ESI-MS assays confirmed the identities of the isolates, but both yielded DNA signatures distinct from that of the clinical isolate.We were successful in isolating several Francisella strains from natural warm springs; however, none appeared to genetically match the original 2001 clinical isolate.This work highlights the presence of viable, potentially pathogenic Franscisella species living in the unique environmental niche of natural warm springs.