Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.