Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family. In the plasmid-encoded Ysc-Yop type III secretion system of human pathogenicYersiniaspecies, YscX is a secreted substrate while YscY is its non-secreted cognate chaperone. Critically, neither anyscXnoryscYnull mutant ofYersiniais capable of type III secretion. In this study, we show that the genetic equivalents of these proteins produced as components of other type III secretion systems ofPseudomonas aeruginosa(PscX and PscY),Aeromonasspecies (AscX and AscY),Vibriospecies (VscX and VscY), andPhotorhabdus luminescens(SctX and SctY) all possess an ability to interact with its native cognate partner and also establish cross-reciprocal binding to non-cognate partners as judged by a yeast two-hybrid assay. Moreover, a yeast three-hybrid assay also revealed that these heterodimeric complexes could maintain an interaction with YscV family members, a core membrane component of all type III secretion systems. Despite maintaining these molecular interactions, only expression of the nativeyscXin the near full-lengthyscXdeletion and nativeyscYin the near full-lengthyscYdeletion were able to complement for their general substrate secretion defects. Hence, YscX and YscY must have co-evolved to confer an important function specifically critical forYersiniatype III secretion.