The chaperonin GroEL and its co-factor GroES promote protein folding in an ATP-dependent manner and are known to have an influence on adaptation to diverse stress conditions. The chaperonin complex includes GroEL, that forms a barrel-like oligomer, and GroES that forms the lid. In most eubacteria the GroESL chaperonin is encoded by a single-copy bicistronic operon that includes the groES and groEL genes. Comparative analysis of cyanobacterial genomes showed that the GroESL chaperonin genes were duplicated at least twice during the evolution of heterocystous, filamentous cyanobacteria. Here we study the functional diversification of groEL/groES in the multi-seriate filament forming cyanobacterium Chlorogloeopsis fritschii PCC 6912. The genome of C. fritschii encodes two groESL operons (groESL1, groESL2) and a monocistronic groEL gene (groEL3). A comparison of gene expression under stress conditions shows that groEL1 is upregulated during temperature stress whereas the monocistronic groEL3 is upregulated under light stress. The expression of groEL2 is induced upon nitrogen deficiency during heterocyst differentiation. In addition, expression of groEL1 is localized in a distinct pattern under diazotrophy. To establish the GroEL-GroES specificity, we tested for protein interactions between the chaperonin subunits in vivo. Subunits encoded in the two operons form hybrid complexes, whereas GroEL3 subunits are not forming oligomers nor interact with any of the two co-chaperonins. Interaction between GroES2 and GroEL2 could not be documented, indicating that the GroESL2 operon does not encode a functional chaperonin complex. Experiments of functional complementation in E. coli confirm that groESL1 can substitute the native operon. Furthermore, groEL2 could complement the native groEL only in combination with groES1. GroEL3 was not functionally complement in any combination of co-chaperonins. Our results demonstrate that the evolutionary consequences of groEL duplication include specialization as a housekeeping gene of groEL1, subfunctionalization of groEL2 and neofunctionalization of groEL3.