Shigellosis is one of the major causes of morbidity in children with diarrhea in Iran. The aim of this study was to investigate antimicrobial resistance of Shigella flexneri (S. flexneri) strains isolated from clinically diagnosed cases of gastroenteritis and acute diarrhea in Tehran, Iran. Shigella strains were isolated from stool samples of patients who visited the several major hospitals in Tehran. S. flexneri was preliminarily identified by biochemical tests as well as by API20E. Antimicrobial resistance testing was performed according to the standard guidelines of the Clinical and Laboratory Standards Institute. All strains were resistant to streptomycin. More than 96.4% of the strains were resistant to tetracycline and amoxicillin, 89% to co-trimoxsazole, 72.6% to ampicillin, 33.3% to chloramphenicol, 9.5% to kanamycin, 1.2% to cefixime, amikacin and furazolidone. None of the tested isolates were resistant to ceftriaxone, ceftizoxime, ceftazidime, gentamicin, ciprofloxacin, cephalothin, cefotaxime, cephalexine, nalidixic acid and nitrofurantoin. More than 96% of the strains showed multi-drug resistance phenotype. Seventeen resistance patterns were identified among the strains, however the most prevalent phenotype (36.9%) was R6 (streptomycin/amoxicillin/tetracycline/co-trimoxsazole/ampicillin). This study indicates an increase in incidence of multiple drug resistance among the strains of S. flexneri isolated in Tehran, Iran.