Background Diarrheal disease is still a health problem, especially in developing countries, where it is considered one of the foremost causes of death in children, accounting for approximately two million deaths annually worldwide. Enteroinvasive Escherichia coli (EIEC) is known to cause shigellosis-like symptoms in both adults and children. Objectives The aim of this study was to identify EIEC in children with diarrhea using Real-time PCR assays in Shiraz, Iran. Patients and Methods A total of 285 stool samples were collected from patients with diarrhea in Shiraz, in 2012. Diarrheagenic E. coli (DEC) strains were isolated by standard biochemical analysis. We used Real time PCR and PCR to detect the presence of ipaH gene in EIEC. Susceptibility testing to 18 antimicrobial agents was determined by diffusion methods according to clinical and laboratory standards institute (CLSI, 2011) guidelines. Results In total, 285 stool samples were tested in which 49 (17%) were contaminated with E. coli by biochemical tests. In the present study, EIEC was detected in seven (14.3%) children with diarrhea. Of seven patients, five children were younger than 12 months and two were 13-24 months old. In total, four EIEC strains were isolated from watery diarrhea and three EIEC strains from bloody diarrhea. In the present study, EIEC strain exhibited high frequency of drug resistance to penicillin (100%), trimethoprim/sulfamethoxazole (71%) and tetracycline (71%). Conclusions We reported the first study performed in Shiraz to identify EIEC intestinal pathogens in children with diarrhea. This type of pathogen should be considered when designing preventive strategies for children in Iran.