Shigella is among the leading bacterial pathogens responsible for diarrheal disease causing > 200,000 deaths per year. Shigella flexneri, one of four Shigella species with 15 defined serotypes/subgroups, is the predominant Shigella species recovered from pediatric shigellosis in low- and middle- income countries (LMICs). Despite significant public health efforts, Shigella continues to be a major health concern. Shigella vaccine development has been hindered largely by the antigenic and genomic diversity. A quadrivalent Shigella vaccine including S. flexneri 2a (Sf2a), S. flexneri 3a (Sf3a), S. flexneri 6 (Sf6), and S. sonnei would provide 84.7% coverage across LMICS, providing protection for children in LMICs as well as travelers and military personnel. Of these three S. flexneri serotypes, Sf6 is the most phylogenomically and phenotypically distinct compared to other S. flexneri serotypes. We hypothesize that the distinct genomic content of Sf6 confers serotype-specific host-pathogen interactions. Preliminary results comparing historical clinical (archetype strains) Sf2a strain 2457T, Sf3a strain J17B, and Sf6 strain CCH060 demonstrated that CCH060 contains the greatest amount of unique genomic content, as well as lacking several previously described Shigella virulence factors. Phenotypic analysis revealed that there is variation in the expression and production of the virulence Ipa proteins and reduced invasion by CCH060 in in vitro assays. Utilizing comparative genomics on a larger collection of Sf6 genomes from diverse geographic locations, we identified similarity among Sf6 strains, despite disparate collection time frames and global locations. The unique genetic properties shared among Sf6, included potential virulence factors (Type II Secretion System) and genomic variation in the Type III Secretion System. We confirmed that geographically representative Sf6 strains from Africa and Asia demonstrated reduced invasion phenotype and Ipa effector secretion. Overexpression of Ipa proteins did not rescue the invasion phenotype. Through preliminary genomic and phenotypic comparisons of Sf6 to S. boydii serotypes 2, 4, and 14, we identified several Sf6 unique genes absent from non-Sf6 S. flexneri (Type II Secretion System and intrinsic antimicrobial resistance genes) and the reduced invasion phenotype was conserved in S. boydii. Together these data highlight highly conserved and unique Sf6 genotypic and phenotypic features, not found within other S. flexneri serotypes.