Eighty-six strains of Shigella spp. were isolated during the dry season from stool samples of children under 5 years of age in Ifakara, Tanzania. The epidemiological relationship as well as the antimicrobial susceptibility and mechanisms of resistance to ampicillin, chloramphenicol, and co-trimoxazole were investigated. Four different epidemiological tools, pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic (REP)-PCR, plasmid analysis, and antibiogram, were compared for typing Shigella strains. Seventy-eight (90%) strains were Shigella flexneri and were distributed into four groups, by either PFGE or REP-PCR, with 51, 17, 7, and 3 strains. The four strains of Shigella dysenteriae belonged to the same group, and the four strains of Shigella sonnei were distributed in two groups with three and one strain each. Plasmid analysis showed a high level of heterogeneity among strains belonging to the same PFGE group, while the antibiogram was less discriminative. REP-PCR provided an alternative, rapid, powerful genotyping method for Shigella spp. Overall, antimicrobial susceptibility testing showed a high level of resistance to ampicillin (81.8%), chloramphenicol (72.7%), tetracycline (96.9%), and co-trimoxazole (87.9%). Ampicillin resistance was related to an integronborne OXA-1-type β-lactamase in 85.1% of the cases and to a TEM-1-type β-lactamase in the remaining 14.8%. Resistance to co- trimoxazole was due to the presence of a dhfr Ia gene in all groups except one of S. flexneri, where a dhfr VII gene was found within an integron. Chloramphenicol resistance was associated in every case with positive chloramphenicol acetyltransferase activity. All strains were susceptible to nalidixic acid, ciprofloxacin, ceftazidime, cefotaxime, and cefoxitin. Therefore, these antimicrobial agents may be good alternatives for the treatment of diarrhea caused by Shigella in Tanzania.