This study aims to determine the prevalence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii by three methods of diagnosis (microscopic examination, cultivation and PCR) that were compared to obtain an accurate diagnosis of Entamoeba spp. during amoebiasis. Total (n=150) stool samples related to patients were (n = 100) and healthy controls (n= 50). Clinically diagnosed stool samples (n=100) were collected from patients attending the consultant clinics of different hospitals in Basrah during the period from January 2018 to January 2019. The results showed that 60% of collected samples were positive in a direct microscopic examination. All samples were cultivated on different media; the Brain heart infusion agar showed high efficiency and was the most suitable in cultivating the parasite. Data and results of molecular study were indicated by DNA extraction from stool samples and used in PCR technique with specific primers. This study identifies different infection percentage for the three species. The highest infection in Basrah patients was Entamoeba moshkovskii 15% followed by Entamoeba dispar 10% and Entamoeba histolytica, which was 5%.