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Eubacteria synthesize a full-length (ClpB95) and a N-terminally truncated (ClpB80) version of the ClpB disaggregase owing to the presence of a translation initiation site within the clpB transcript. Why these two isoforms have been evolutionary conserved is poorly understood. Here, we constructed a series of E. coli strains and plasmids allowing production of the ClpB95/ClpB80 pair, ClpB95 alone, or ClpB80 alone from near physiological concentrations to a 6–10-fold excess over normal cellular levels. We found that although overexpressed ClpB95 or ClpB80 can independently restore basal thermotolerance to Δ clpB cells, strains expressing ClpB80 from the clpB chromosomal locus do not exhibit increased resistance to thermal killing at 50 °C relative to clpB null cells. Furthermore, synthesis of physiological levels of ClpB95 is less effective than coordinated expression of ClpB95/ClpB80 in protecting E. coli from thermal killing. These results provide an explanation for the conservation of the two ClpB isoforms in eubacteria and are consistent with the fact that wild type E. coli maintains the ClpB80 to ClpB95 ratio at a nearly constant value of 0.4–0.5 under a variety of stress conditions.