Salmonella enterica subspecies enterica serotype 4,[5],12:i:- is an emerging serovar considered as a monophasic variant of Salmonella enterica serotype Typhimurium. The antigenic and genetic similarity between Salmonella 4,[5],12:i:- and Salmonella Typhimurium suggests that they may behave in a similar way and represent a comparable threat to public health. As serotyping alone does not necessarily provide for identification of Salmonella 4,[5],12:i:- and its differentiation from Salmonella Typhimurium, a method that combines traditional serotyping and a multiplex polymerase chain reaction has been tested on 208 strains serotyped as Salmonella 4,[5],12:i:-, Salmonella Typhimurium, and similar serovars of serogroup B sharing the same phase-1 antigen "i." For 191 strains, the combined method fully confirmed the results provided by traditional serotyping, whereas for 17 strains of Salmonella 4,[5],12:i:- and Salmonella Typhimurium some inconsistencies emerged between the two methods. The combined method resulted in a more accurate and faster identification of these two relevant serovars.