The current research aimed to diagnose Entamoeba histolytica and Entamoeba dispar by the use of nested-PCR. As the study, 100 samples of stool of patients with moderate and severe diarrhea with or without blood in the stool were collected at Tikrit Teaching Hospital, Al-Alam Hospital, and other laboratories in the Al-Alam District (between early October and late June 2025). The age of patients ranged between one month to 40 years with all genders. Diagnosis of the samples was based on morphological and molecular analysis through the Nested Polymerase Chain Reaction technique with the emphasis on the 18S rRNA gene with E. histolytica-specific and E. dispar-specific primer pairs. It was found that the general rate of infection of Entamoeba spp. was 65 percent of the total samples examined under the microscope. Molecular study of the Entamoeba genus showed that the 900-base-pair fragment of the 18S rRNA gene was detected in 88% of the sample analysed whereas the Eh1-Eh2 gene of E.histolytica was detected in 42% of the positive samples and ED-1 and ED-2 genes in E.dispar were found in 27%.