Phytoplankton species can produce resting stages that persist in the sediment over long periods and accomplish important ecological functions. With the aim of investigating the phytoplankton assemblage structure in relation to environmental drivers and human pressures, we analyzed resting stages of diatoms and dinoflagellates, including harmful algal bloom taxa, in the surface sediments of 3 Mediterranean regional areas. Abundance of resting stages was determined by molecular quantitative polymerase chain reaction assay. Multivariate data analysis confirmed that the abundance of resting-stage assemblages seemed related to depth. Regional differences in the composition of the resting-stage assemblages were evident and environmental drivers were correlated with those regional differences. Three main groups of samples were defined according to SST and depth thresholds. Samples from the northern and central Adriatic Sea (average SST < 18°C) formed the richest assemblages, both in terms of abundance and species richness, while deep samples from all other basins (depth > 368 m) were poorer and less diverse than those from shallower sites (depth ≤ 368 m). Resting-stage taxa contributed differently to the 3 groups. Diatom spores and dinoflagellate cysts were the most abundant taxa, but Alexandrium minutum cysts and Ditylum brightwellii spores also accounted for a large share of the overall inter-group compositional distance. The structure of resting-stage assemblages can be regarded as a time- and space-integrated response of a subset of phytoplankton species to environmental conditions, including the physical oceanographic dynamics that favor or prevent sedimentation of resting stages.