The present study aims at the differentiation of Entamoeba histolytica and Entamoeba dispar using nested multiplex PCR after detection of the Entamoeba by conventional parasitological techniques. A total of 490 stool samples were collected from 300 symptomatic patients who were attending the outpatient clinics of Kasr Al-Aini, Abou El Reish and the health centre of the Ministry of Health in Gesr Al-sowais (known as the “Ancylostoma Unit”) as well as 190 asymptomatic people performing routine check-up at the French Kasr Al-Aini Hospital. Stool samples were examined by direct wet mount, ether sedimentation concentration technique and after trichrome staining. E. histolytica/dispar was detected by direct microscopic examination in 39 stool sample which represent 7.9% of the studied population. When nested multiplex PCR was applied to the samples proved to be positive by microscopic examination, all of these samples proved to belong to Entamoeba species. Infection with E. histolytica only was 69.2% (n=27), E. dispar only in 10.3% (n=4), mixed infection by both E. histolytica and E. dispar was 15.4% (n=6) and 5.1% (n=2) of the Entamoeba spp. samples belonged to neither histolytica nor dispar. The present study highlighted the effectiveness of nested multiplex PCR as a technique for species specific detection and differentiation of E. histolytica and E. dispar DNA in stool samples.