Salmonella enterica subsp. enterica 4,[5],12:i:- is one of the most prevalent serovars associated with human infections worldwide. Two multidrug-resistant clones, designated Spanish and European clones, are recognized as having importance for public health and are subject to control measures in the European Union. In this study, 23 clinical isolates belonging to the Spanish clone were characterized by multilocus sequence typing, multiple-locus variable number tandem repeat analysis (MLVA), PCR amplification and sequencing, and a DNA microarray targeting 263 genes, in order to provide new insights into their origins and further evolution. The derived data were compared with information available from other studies for S. 4,[5],12:i:- isolates of both the Spanish and the European clones, to identify differential molecular markers which could be potentially used as surveillance tools in the control of dissemination of this serovar. The isolates analyzed were assigned to sequence type 19 and to 17 MLVA patterns, with 3-13-16-NA-311 being the most prevalent. Highly similar virulence, metabolic, and prophage-associated gene profiles were identified, but DNA mobility markers distinguished five genotypes. Two types of deletions, caused by insertion of IS26, presumably donated by pUO-STmR/RV1-like plasmids typically found in the Spanish clone, affected the fljAB operon and surrounding DNA. The Spanish and European clones differ in sequence type, MLVA patterns, gene repertoire, and fljAB deletion type. The observed variability supports an independent evolution of the two successful monophasic clones from different Salmonella enterica serovar Typhimurium ancestors and can be taken into consideration for epidemiological surveillance.