This study was carried out to differentiate, genetically between Shigella flexneri strains isolated from river Nile water at Sohag governorate. The antibiotic resistance was targeted in this study as well. The antibiotic susceptibility of 11 Shigella flexneri strains was done using 10 different antibiotics. The genetic correlation of these strains was performed by using ERIC and ITS based PCR techniques. Eleven Shigella flexneri, strains were isolated from river Nile water in 7 areas along Sohag governorate. The ratio of S. flexneri in water samples comparing with total bacterial count was also recorded. The antibiotics susceptibility test showed that 100% of strains are resistant to Rifamycin, Cefalexin, and Erythromycin, whereas 91%, 82%, 54.5%, 45.5%, 36.5%, and 27.3% were resistant to Ceftazidim, Cefepim, Cefoxitin, Ertapenem, E-Moxiclav, and  Norfluxacin respectively. The lowest strains percentages were resistant to Ciprofluxacin (18.2% of the strains). By using ERIC-PCR, nine different patterns were observed among the 11 isolates. The pattern was distinct for each isolate, except 3 isolates were fully identical in their profile. ERIC-PCR typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. ITS-PCR did not differentiate between the strains, which separated in only two groups, since it produced few bands profile, identical in most of tested strains. The strains isolated from Sohag city are joint factor with 3 strains isolated from 3 different regions in Sohag governorate representing 50% of the studied areas, because Sohag city is the center of the governorate, and includes the central hospitals receipt shigellosis cases from the other regions. This should alarm the officials to enhance the hygiene process and sewage control, to prevent contamination of drinking water with sewage.