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Typhoid fever caused by Salmonella enterica serovar Typhi has become a serious public health issue due to the development of resistance worldwide especially in developing countries including Pakistan. The aim of this study was to assess the antimicrobial susceptibility pattern of Salmonella Typhi isolated from human blood samples and detect genes by using PCR technique. Total 400 blood samples of typhoid confirm patients were cultured on Salmonella-Shigella agar to isolate Salmonella Typhi. The bacterium was identified through biochemical tests and susceptibility was checked against ten different antibiotics by using disk diffusion method. PCR technique was used to detect different genes after extraction of DNA using phenol-chloroform method. Salmonella typhi (27%) was isolated from blood samples of typhoid patients. Among these, 41% were resistant to ciprofloxacin followed by 34% of ceftriaxone and 25% of streptomycin. Isolated strains were positive for the presence of gyrase A, CMY-2, spvC and InvA genes detected through PCR using specific primers. This study concluded that Salmonella typhi is developing resistance to antimicrobial agents due to the transfer of virulence and resistant genes which may lead to the failure in treatment of typhoid patients in Pakistan. [ABSTRACT FROM AUTHOR]