A new benthic sensor for the differentiation of algal groups in mixed microphytobenthos populations in situ with high spatial and temporal resolution has been developed. The differentiation is based on the concept that five spectral excitation ranges can be used to determine groups of microalgae in situ within a few seconds. As sediments contain a lot of yellow substances, which can effect the optical differentiation of the algae, the device is additionally equipped with a correcting UV-LED for yellow substances. Results from the differentiation on microphytobenthos populations and their migrating behaviour in the field and in mesocosm experiments are compared with algal biomass and pigment estimations. The usefulness of this non-retrospect approach is discussed critically in view of its potential role for ground truth investigations on the large-scale spatial and temporal variation of algal populations in sediments.