Mycobacterium smegmatis contains three chaperonin (cpn60) genes homologous to the Escherichia coli groEL gene. One of these (cpn60.1) is required for biofilm formation, but is nonessential, whereas a second (cpn60.2) is essential. Mycobacterium smegmatis is unique among Mycobacteria in having a third chaperonin gene, cpn60.3. The cpn60.1 gene has a gene upstream (cpn10) that is homologous to the gene for the E. coli co-chaperonin GroES. Phylogenetic analysis of the mycobacterial homologues suggests that early gene duplication and sequence divergence gave rise to the cpn60.1 and cpn60.2 genes found in all Mycobacteria species, while cpn60.3 appears to have been acquired by horizontal gene transfer. Here, we show that cpn60.2 and cpn10 are expressed more strongly than cpn60.1, while cpn60.3 shows very low levels of expression. The expression of all the genes, except cpn60.3, is significantly induced by heat shock, but much less so by other stresses. We mapped mRNA 5′-ends for the cpn10 and cpn60.1 genes, and measured the promoter activity of the upstream regions of both genes. The results show that the mRNA for this operon is cleaved between the cpn10 and cpn60.1 genes. These results are consistent with the evolution of a distinct function for the cpn60.1 gene.