This study examined the ability of the Photochemical Reflectance Index (PRI) to track seasonal variations in carotenoid pigments and photosynthetic activity of mature evergreen chaparral shrubs. Our results confirm that PRI scales with photosystem two (PSII) photochemical efficiency across species and seasons, as demonstrated by PRI’s strong correlation with de-epoxidized (photoprotective) xanthophyll cycle pigment levels (normalized to chlorophyll) and with the chlorophyll fluorescence index, ΔF/Fm’. PRI and carotenoid pigment levels (de-epoxidized xanthophyll cycle pigments normalized to chlorophyll or total carotenoid pigments normalized to chlorophyll) were correlated with seasonal fluctuations in midday net CO2 uptake of top-canopy leaves. By contrast, chlorophyll levels (as measured by the Chlorophyll Index) were not as strongly linked to photosynthetic activity, particularly when all species were considered together. Likewise, the Normalized Difference Vegetation Index (NDVI, an index of canopy greenness) did not correlate with net CO2 uptake. Canopy NDVI also did not correlate with canopy PRI, demonstrating that these indices were largely independent over the temporal and spatial scales of this study. Together, these patterns provide evidence for coordinated regulation of carotenoid pigments, PSII electron transport, and carboxylation across seasons and indicate that physiological adjustments are more important than structural ones in modifying CO2-fixation capacity during periods of photosynthetic down-regulation for these evergreen species. The strong correlation between PRI of whole canopies and PRI of top-canopy leaves suggests that the canopy can be treated as a “big leaf” in terms of this reflectance index and that PRI can be used in “scalable” models. This along with the links between carotenoid pigments, PSII photochemical efficiency and carboxylation across species and seasons supports the use of optical assays of pigment levels and PSII activity in CO2 flux models to derive photosynthetic rates.