Bacillary diarrhea caused by Shigella flexneri is mediated by various virulence factors which make it the leading agent of diarrhea in developing countries. Previously, a high prevalence of S. flexneri, associated with diarrhea has been reported in Pakistan but no data is available on their virulence profile. The present study reporting for the first time analysis of various virulence factors among S. flexneri serotypes isolated from clinical (diarrheal stool) and non-clinical (retail raw foods and drinking water) sources. A total of 199 S. flexneri (clinical: 155, raw foods: 22, water: 22) belonging to various serotypes were subjected to virulence genes detection and virulence profiling. The most frequent virulence gene was found to be ipaH (100%), followed by sat (98%), ial (71.3%), set1B (65.8%) and set1A (38.7%). A high level of virulence was detected in serotype 2b as compared to other serotypes as 32.3% of all serotype 2b have the entire set of five virulence genes including ipaH (100%), ial (100%), sat (37.7%), set1A (89.3%), and set1B (100%). Seven different virulence gene profiles (V1 - V7) were detected and the most frequently observed to be V1 (ipaH+, ial+, sat+, set1A+, set1B+) followed by V3 (ipaH+, ial+, sat+, set1B+). The predominant virulence gene pattern in serotype 2b isolated from clinical and non-clinical samples were V1 and V3. Furthermore, about 32% strains belongs to serotype 2b contain the complete set of five virulence genes isolated from patients with high disease severity. In conclusion, the current finding revealed for the first times that serotype 2b was the most virulent strains in both clinical and non-clinical samples in Pakistan. In addition, the virulence of serotype 2b was well correlated with high disease severity.