Background: The mycobacterial PE_PGRS protein family is present only in pathogenic strains of the genus mycobacterium, such asMtband members of the MTB complex, suggesting a likely important role of this family in pathogenesis. Their PGRS domains are highly polymorphic and have been suggested to cause antigenic variations and facilitate pathogen survival. The availability of AlphaFold2.0 offered us a unique opportunity to better understand structural and functional properties of these domains and a role of polymorphism inMtbevolution and dissemination. Methods: We made extensive use of AlphaFold2.0 computations and coupled them with sequence distribution phylogenetic and frequency analyses, and antigenic predictions. Results: Modeling of several polymorphic forms of PE_PGRS33, the prototype of the PE_PGRS family and sequence analyses allowed us to predict the structural impact of mutations/deletions/insertions present in the most frequent variants. These analyses well correlate with the observed frequency and with the phenotypic features of the described variants. Conclusions: Here, we provide a thorough description of structural impacts of the observed polymorphism of PE_PGRS33 protein and we correlate predicted structures to the known fitness of strains containing specific variants. Finally, we also identify protein variants associated with bacterial evolution, showing sophisticated modifications likely endowed with a gain-of-function role during bacterial evolution.