Multilocus sequence typing (MLST) based on the 16S RNA, pduF, glnA, and manB genes was developed for Salmonella, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231 Salmonella isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of Salmonella is better than that of serotyping and/or PFGE typing. manB-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose manB gene sequences were analyzed. The G+C contents and Splitstree analysis of the manB, glnA, and pduF genes of Salmonella indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.