We take an in vivo fluorescence approach to investigate photosynthetic adaptation by ecologically divergent members of the A/B clade of the hot spring cyanobacterium Synechococcus, the most thermotolerant of which defines the upper thermal limit for photosynthesis. During Synechococcus diversification, both photosystem II and the light-harvesting phycobilisome have evolved greater thermostability as the group has invaded higher temperature habitats, particularly for the most thermotolerant lineage. This enhanced function at higher temperatures has come at the cost of reduced performance at lower temperatures, and these trade-offs contribute to niche specialization in the clade. Molecular evolutionary analyses revealed specific adaptive protein changes in the most thermotolerant lineage. Our study advances our understanding of the origins of Synechococcus diversity.