In this study we describe a multiplex PCR assay for the detection of six clinically relevant antibiotic resistance genes of salmonella typhi stains isolated from different South East Asian countries. Conditions were optimized to amplify fragments of 798 bp tem-1 (encoding resistance to trimethoprim),308 bp dps-1 and  dps-2  (sulfonamide resistance), 293 bp cat I (chloramphenicol  resistance, 678 bp tet A (tetracycline resistance), and dhfr-7 (trimethoprim-sulfamethoxazole resistance)), simultaneously in two PCR amplification. The multiplex PCR assay was evaluated on A total of 79 S.typhi strains from sporadic cases of typhoid fever, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. PCR for the detection of antimicrobial resistance genes after spiking in blood culture in different incubation times was investigated. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.