Background: Shigella flexneriis an emerging threat in low socioeconomic countries including Pakistan. No previous data is available on the association betweenS. flexneriserotypes and antimicrobial resistance in Pakistan. Objectives: The objective of the present study was to assess the association between serotypes and antimicrobial resistance patterns amongS. flexneriisolated from clinical and nonclinical samples. Methods: A total of 199S. flexneriisolates were subjected to molecular serotyping and antibiotic resistance. Results: The most prevalentS. flexneriserotype was 2b (38%) followed by 1b (24%), 7a (20%), 2a (11%), 1d (5%) and Y (2%). The phylogenetic reconstruction showed 12 clades among which the clades II, III, V, VIII, IX and XI have consisted of serotypes that were found both in human population and environment samples. A high level of multidrug resistance (MDR) was observed in serotype 2b (37.68%) followed by 1b (19.5%) and 7a (19.5%), 2a (11.5%), 1d (5%) and Y (2%). All isolates of serotype 2b showed high level of resistance to amoxicillin/clavulanic acid (100%) followed by quinolone (74.6%) and trimethoprim-sulfamethoxazole (54.6%). Interestingly, none of the serotype was resistant to piperacillin-tazobactam, imipenem and amikacin. The most frequently detected resistance genes among serotype 2b werebla(OXA)(100%) followed byqnrS(88%),cat(81%) andsul2(63%). Conclusion: The most frequentS. flexneriserotype was 2b while 1d and Y was first time reported in Pakistan. High frequency of MDR serotypes ofS. flexneriis a serious threat in diarrhea endemic regions and thus require urgent strategies for its continuous monitoring and prevention.