Background  Entamoeba histolytica is a unicellular protozoon that conceder as a common cause of dysentery in human especially in the children under 6 years old. This may be transmitted via the ingestion of cystic stage of the parasite.  Objectives  This study aimed to investigate the total rate of Entamoeba histolytica infection in children admitted to the Pediatric Teaching Hospital in Sulaimani City, and considering some other aspects such as culturing and detecting subtypes using polymerase chain reaction (PCR).  Patients and Methods This epidemiologic study includes 1005 stool samples collected from children aged 6 months to 6 years old from the 1st of Jun to the 30th of October 2014. Each stool sample was examined and identified by direct wet mount method using normal physiological saline and iodine solution, followed by culturing and isolation of the parasite for 14 days to elongate life span and then genomic amplification applied from the cultured Entamoeba histolytica by appropriate PCR based method. Results The result of rapid kit occult blood combined with microscopic wet mount (iodine) showed that the infection with E. histolytica was (12.9%), in which the rate in male (14.18%) was higher than that female (11.42%), with no significant difference between both genders. cultured E. histolytica showed a great ability of this parasite to grow, with detection of the parasite under light microscope (40X). Then two standard kits were used specific primer (1147, F&R), and thus about 1147bp PCR produced. Then, nested PCR was performed for the same PCR products two different primers (246 F&R) which resulted in fragments of approximately 246bp. This fragment coding for the 16S SSUrDNA gene of HM-1IMSS 16s E. histolytica confirms the previous PCR amplification finding. The results of sequence methods identified E. histolytica subtype 1 from PCR positive samples by sequencing alignment (99%) similarity with accession number KB823016.  Conclusion The prevalence rate of infection with E. histolytica in Pediatric Teaching Hospital in Sulaimani City was (12.9%). Beef extract medium for culturing of E. histolytica showed a great ability for growing of the parasite cystic form. E. histolytica subtype1 can be detected by sequencing method for the first time in molecular genotype of human E .histolytica in Sulaimani city.