Although Salmonella remains one of the leading causes of foodborne illnesses in the United States, the Salmonella enterica serovars and genetic types associated with most infections appear to fluctuate over time. Recently, the Center for Disease Control and Prevention (CDC) has reported an increase in cases of salmonellosis caused by Salmonella 4,[5],12:i:−. Similarly, this unusual Salmonella serovar has been isolated from cattle and poultry in the state of Georgia. We examined the genetic relatedness of Salmonella 4,[5],12:i:−, isolated from several different poultry companies and dairy farms in Georgia, by pulsed-field gel electrophoresis (PFGE). Several Salmonella 4,[5],12:i:− isolates had PFGE patterns identical or similar to PFGE patterns of Salmonella Typhimurium isolated from numerous animal sources. We identified distinct PFGE patterns for Salmonella 4,[5],12:i:− and matching Salmonella Typhimurium PFGE patterns, identifying four “distinct” strains. We focused a more specific analysis on the poultry Salmonella 4,[5],12:i:− and Salmonella Typhimurium isolates and found that of these Salmonella 4,[5],12:i:− isolates, 32% lacked the entire phase 2 antigen gene, fljB; 61% contained partial deletion(s); and 4% had partial deletion(s) in fljB and an adjacent gene hin, 5′ to fljB. Thirteen percent contained smaller deletions or point mutations not identified by our DNA probes. The Salmonella 4,[5],12:i:− isolates were positive for several genes present in the Salmonella Typhimurium, including lpfE (100%), sseI (96%), and spvC (93%). Genetic analysis indicates independent, spontaneous mutations in fljB in at least four distinct Salmonella Typhimurium strains of animal origin circulating in nature.