Identifying the genetic changes underlying adaptive diversification is a fundamental goal of evolutionary biology.  We are taking an integrative approach to elucidate the mechanisms of temperature adaptation in related but ecologically divergent lineages of the thermophilic cyanobacterium Synechococcus, the most tolerant of which defines the thermal limit for photoautotrophy.  Here, we tested whether adaptive evolution of the Calvin cycle enzyme RubisCO has contributed to Synechococcus diversification.  CD thermal scans revealed differences in evolved thermostability of this enzyme, including a substantial enhancement of stability for the most thermotolerant lineage of Synechococcus.  We next used site-directed mutagenesis to address the genetic basis of evolved increases in RubisCO thermostability.  Two replacements of alanine by isoleucine at highly conserved sites in the RubisCO large subunit were found to be responsible for much of the adaptive gain in stability observed for the most thermotolerant lineage.  Our working model is that additional methylene groups contributed by these residues may synergistically enhance thermal stability by facilitating tighter packing and van der Waals interactions within the protein interior. The evolution of enhanced thermostability has also come with the expected cost of reduced catalytic performance at lower temperatures, a biochemical trade-off that mirrors fitness trade-offs among strains at their respective physiological extremes.