Shigella flexneri is endemic in most developing countries and responsible for the highest mortality rate among the Shigella species. The attenuated serotype Y S. flexneri strain SFL124 has been used as the parental strain for the development of recombinant vaccines expressing multiple O-antigen structures. During the development of one such multivalent vaccine, a region of gtrII homology was found in SFL124. Sequencing and analysis of this region revealed the presence of an insertion element interrupted serotype 2a serotype-conversion locus in the serotype Y vaccine strain SFL124. The data presented suggests that SFL124 has derived from a serotype 2a background.