Abstract: The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.