Multilocus variable number tandem repeat (VNTR) analysis (MLVA) is a new typing method with several advantages compared to other methods. Dissemination of Shigella is highly significant in developing countries. Whilst Shigella is becoming increasingly important as an etiologic agent of pediatric shigellosis in Iran, little is known about the genetic diversity of the local strains. Therefore, the aim of this study was to describe the genetic diversity of Shigella species isolated from pediatric patients in Tehran, Iran. A total of 53 Shigella isolates were obtained from 1070 patients with diarrhea (less than 12 years of age). All isolates were identified by routine biochemical and serological tests. The confirmed Shigella isolates were further serogrouped (by the slide agglutination) using slide agglutination method. MLVA assay with the seven loci resolved 53 Shigella isolates into 36 different genotypes. Almost all the isolates were classified into five clonal complexes. Furthermore, our MLVA assay could effectively distinguish the four Shigella species. This study has provided valuable insights into the genetic heterogeneity of Shigella species in Tehran, Iran. Our findings can be helpful for further epidemiological surveillance of Shigella species in this country in the future.