Abstract: Background and Aim: In most part of the world detection of cysts and trophozoites of Entamoeba is based on morphological structure of this species in stool sample by microscopy. However, microscopic examination is unable to distinguish between similar morphological protozoa such as Entamoeba histolytica and Entamoeba dispar. A simple and cost-effective method is needed in medical laboratories for detection and differentiation of these two species. Materials and Methods: Stool samples of patients who were referred from health care centers were examined by direct microscopy and trichrome stain. Polymerase chain reaction (PCR) utilizing *pEd30F and pEd21AS primers from Peroxiredoxin gene, was used for differentiation of E. histolytica and E. dispar. Genomic DNA from samples was amplified by these primers. The fragment under 100 bp was related to E. histolytica and in contrast the fragment above the 100 bp was related to E. dispar. Results: In this study from 22 microscopic positive samples, E. histolytica was observed only in one patient and E. dispar was detected in the other 21 samples. Discussion: The result of this study indicate that the PCR reaction could amplify E. dispar and E. histolytica with just one primer pair and this is a cost-effective method for distinguishing between these two species. Keywords: Entamoeba histolytica, Entamoeba dispar, Peroxiredoxin Gene. *Corresponding Author: Ehsan Nazemalhoseini Mojarad National Research Department of Foodborne and Diarrhea Diseases, Research Center for Gastroenterology and Liver Diseases, Taleghani General Hospital, Shahid Beheshti Medical University, Tehran, Iran. Email: ehsanmojarad@gmail.com

Keywords: Entamoeba histolytica, Entamoeba dispar, Peroxiredoxin Gene.

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