Shigella flexneri is among the common causes of bacillary dysentery in children, especially in developing countries. The intracellular invasion and replication of Shigella within intestinal epithelial cells are regulated by a number of virulence-associated genes, the most important of which are virF and ial, situated on a large virulence plasmid. One hundred stool specimens were obtained from pediatric patients suffering from acute diarrhea attended Al-Diwaniyah, Iraq. Shigella flexneri was confirmed through microbiological and biochemical tests among a portion of the isolates from which genomic DNA was extracted for molecular studies. PCRs were carried out as described for the virF and ial genes using specific primers. The virF gene was detected in 40% isolates, and the ial gene in 30% of isolates, and the combination of both genes was noted in few percentages of isolates. These results reveal significant genetic diversity in circulating strains and emphasize the potential of molecular screening for highly virulent isolates.The aim of the study was to detect the presence of virF and ial genes in Shigella flexneri isolates to assess genetic variability and identify potentially more virulent strains. Comprehension of the distribution of invasion associated genes may strengthen epidemiological monitoring and might have potential implications to improve diagnostic methods and preventive measures for pediatric shigellosis.