The prevalence, among human clinical cases, of Salmonella enterica serotype 4,5,12: i:-, a serotype antigenically similar to Salmonella enterica serotype Typhimurium but lacking second-phase flagellar antigens, has increased considerably over the last 10 years. To probe the evolution and ecology of this emerging serotype, we characterized 190 Salmonella isolates initially classified as Salmonella serotypes 4,5,12: i:- (n = 90) and Typhimurium (n = 100) and obtained from various sources in the United States and Spain. These isolates were characterized into six sequence types (determined by multilocus sequence typing [MLST]) and 79 pulsed-field gel electrophoresis types. The majority of Salmonella serotype 4,5,12: i:- and Typhimurium isolates (85 and 84 isolates, respectively) represented a single MLST type. Existing genome information revealed different genome deletions (which included genes responsible for phase 2 flagellum expression) in four Spanish Salmonella serotype 4,5,12: i:- isolates and one U. S. Salmonella serotype 4,5,12: i:- isolate. Fifty-nine isolates of both serotypes, representing different sources and geographical locations as well as different molecular subtypes, were thus screened for the presence of six genes and one specific region, all of which were previously found to show variable presence among Salmonella serotype 4,5,12: i:- and Typhimurium strains. All Salmonella serotype 4,5,12: i:- isolates lacked the phase 2 flagella genes fljA and fljB, which were present in all Salmonella serotype Typhimurium isolates. While all Spanish Salmonella serotype 4,5,12: i:- isolates carried the same deletion surrounding fljAB, all but two U. S. isolates showed a different genomic deletion; the two atypical U. S. isolates represented the "Spanish" deletion genotype and a unique deletion genotype. Salmonella serotype 4,5,12: i:- thus appears to represent at least two common clones, which cannot easily be differentiated with standard diagnostic procedures.