The use of pulse-amplitude-modulated fluorometry (PAM) and rapid light curves (RLC) were evaluated for monitoring the physiological condition of the seagrass, Thalassia testudinum, at the landscape scale in Florida Bay, USA. PAM fluorometry provides rapid, non-invasive, and quantitative physiological information on the state of photosynthesis. Yet, previous studies of effective and maximum quantum yields have shown that problems arise when expanding measurements from the organismal scale to the landscape scale, mainly due to temporal and irradiance-induced changes in photophysiology. Here, the magnitude of diurnal and spatial variation of photosynthetic characteristics among 10 sample basins and between two sample years was investigated using RLCs. Because RLCs measure effective quantum yields over a range of changing actinic irradiances, we hypothesized that the response parameters might be less sensitive to diurnal light history effects. Our results indicate that the RLC parameters, alpha and ETRmax, significantly changed diurnally, as was previously found for both maximum and effective quantum yields, but the diurnal patterns were variable among the 10 basins. Both among-basin and between-year comparisons were confounded by diurnal variation and statistical analyses comparing morning, mid-day, and afternoon time periods were unable to definitively discern which time of day was best suited for assessing the relative photophysiological status of T. testudinum. However, pooling RLC data at the basin scale revealed among-basin differences and landscape scale trends that were consistent with basin-level morphometric variation in this seagrass. Thus, PAM fluorometry may be useful as a landscape scale monitoring tool within certain constraints. When using this approach over large spatial and temporal scales, diurnal variability must be considered.