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Background: Diarrheal illnesses caused by Salmonella and Shigella spp remain a serious public health problem in industrializing countries, and are still an important cause of morbidity and mortality in developed as well as undeveloped countries. Rapid detection of agents that cause diarrhea may help prevent occurrence of outbreaks. Objectives: The aim of this study was to compare conventional biochemical tests, namely API 20E strip system and Polymerase Chain Reaction (PCR) for identification of Salmonella enterica and Shigella spp. isolated from stool samples of patients with diarrhea. Materials and Methods: Stool samples were collected from patients with diarrhea during a three-year period (2009)(2010)(2011)(2012). Conventional biochemical tests were used for identification of Shigella spp. and Salmonella enterica isolates. API 20E strips and PCR methods with oligonucleotide primers specific for ipaH of Shigella genus and hilA of Salmonella enterica were used to confirm the identity of the isolates. Results: Of the 81 suspected S. enterica and 112 Shigella spp. identified by conventional biochemical tests, 77 and 105 were identified as S. enterica and Shigella spp. by API 20E, respectively. All of the isolates were assigned to bacterial species with 99.9% probability value. All of the 81 suspected Salmonella enterica isolates produced 784 bp amplification bands of hilA gene. Among the 112 Shigella isolates confirmed by PCR, 90 (80.35%) were positive for wbgZ and 14 (12.5%) were positive for rfc genes indicative of S. sonnei and S. flexneri , respectively. Conclusions: In conclusion, the results of this study suggest that the PCR amplification of hilA and ipaH is a promising method for identification of Salmonella enterica and Shigella spp. The outcomes of this study can help towards more accurate and easy screening of large population of patients with Salmonella enterica and Shigella spp infections.