Entamoeba histolytica infects the large intestine of humans, causing a spectrum of clinical appearances ranging from asymptomatic colonization to severe intestinal and extra-intestinal disease. The parasite is identical microscopically to commensal nonpathogenic amoeba. To detect the pathogenic Entamoeba and estimate the precise prevalence of the parasite among the symptomatic pediatric population using molecular techniques. 323 fecal samples were collected from symptomatic children admitted to Sulaimani Pediatric Teaching Hospital, Sulaimaniyah Province, Iraq, from June to October 2021. A structured, validated questionnaire was prepared and used to report participants' gender, residency, and drinking water source. Then, stool samples were microscopically examined, and the positive samples were submitted to molecular analysis by amplifying the 18s rRNA gene using nested PCR to differentiate E. histolytica from other nonpathogenic Entamoeba. Finally, gene sequences were done to confirm the species. Microscopically, 58 positive samples represented Entamoeba species infection rate of 18% among symptomatic patients. However, only 18 samples were positive for E. histolytica based on molecular methods, which accounts for 31% of the positive by microscopy and 5.6% among the 323 symptomatic populations. NCBI, available in their database, gives the gene sequence and accession number. Patients' sociodemographic data and water sources were directly related to the infection rate. Classical microscopic examination provides a misleading profile about the prevalence of E. histolytica in an endemic region that might lead to unnecessary treatments and a lack of appropriate management for patients.