OBJECTIVES: The objective of this study was to evaluate the present status of amoebiasis in Thi-Qar Province in southern Iraq, and to determine the presence of <i>Entamoeba histolytica</i> and <i>Entamoeba dispar</i> with nested and real-time polymerase chain reaction (PCR).METHODS: Epidemiological data were obtained from the public health department of the Thi-Qar Health Office (2015-2020). Eighty stool samples were also randomly collected from patients ≤12 year of age with diarrhea at 2 hospitals between the beginning of February 2020 and the end of October 2020. These samples were selected after microscopy to identify the <i>18S rRNA</i> gene in <i>Entamoeba</i> DNA.RESULTS: Of the 341,554 cases of intestinal parasitic infections, 38,004 (11.1%) individuals were recorded as having amoebiasis, which accounted for the highest proportion of infections in 2015 (26.1%) and the lowest in 2020 (8.1%). Amoebiasis was distributed among all age groups, with the age group of 5-14 years accounting for the highest proportion (27.3%). In molecular testing, 42 (52.5%) out of 80 samples were positive for the <i>18S rRNA</i> gene (888 bp). Using nested PCR, <i>E. histolytica</i> (439 bp) was detected in 25 (31.3%) samples and <i>E. dispar</i> (174 bp) in 14 (17.5%), while using real-time PCR, <i>E. histolytica</i> and <i>E. dispar</i> were detected in 28 (35.0%) and 15 (18.8%) samples, respectively.CONCLUSIONS: Epidemiological data confirmed that amoebiasis is endemic in this province, and is not limited to certain months. Our study confirms the applicability of molecular identification to detect pathogenic and non-pathogenic <i>Entamoeba</i> to prescribe the appropriate drug.