Many symbioses between bacteria and insects resulted from ancient infections followed by strict vertical transmission within host lineages. The strong bottlenecks under which this transmission occurs promote the neutral fixation of slightly deleterious mutations by genetic drift. As predicted by Muller's ratchet, this fixation will drive endosymbiotic bacteria through an irreversible dynamics of fitness loss. The chaperonin GroEL has been proposed as a compensatory mechanism whereby endosymbiotic bacteria of aphids persist. Here, we show that endosymbiotic bacteria of insects from two phylogenetically very distant bacterial phyla have fixed amino acid substitutions by positive selection in functionally important GroEL regions involved in either GroES/peptide binding or in the en bloc movement of the GroEL apical domain. These results, together with the high levels of constitutive expression of GroEL in these endosymbionts, provide valuable insights into the evolution of a molecular mechanism responsible for the maintenance of the symbiotic lifestyle.