We describe a novel, field-deployable hyperspectral imaging system, called Hypersub, that allows noninvasive in situ mapping of the microphytobenthos (MPB) biomass distribution with a high spatial (sub-millimeter) and temporal (minutes) resolution over areas of 1 × 1 m. The biomass is derived from a log-transformed and near-infrared corrected reflectance hyperspectral index, which exhibits a linear relationship (R2 > 0.97) with the chlorophyll a (Chl a) concentration in the euphotic zone of the sediment and depends on the sediment grain size. Deployments of the system revealed that due to factors such as sediment topography, bioturbation, and grazing, the distribution of MPB in intertidal sediments is remarkably heterogeneous, with Chl a concentrations varying laterally by up to 400% of the average value over a distance of 1 cm. Furthermore, due to tidal cycling and diel light variability, MPB concentrations in the top 1 mm of sediments are very dynamic, changing by 40–80% over a few hours due to vertical migration. We argue that the high-resolution hyperspectral imaging method overcomes the inadequate resolution of traditional methods based on sedimentary Chl a extraction, and thus helps improve our understanding of the processes that control benthic primary production in coastal sediments.