The north-eastern Aegean Sea (NEA) is a marine system of high hydrological complexity, principally induced by the inflow and subsequent advection of the low salinity (<30) Black Sea water (BSW). This water mass occupies the upper layer (~0–20 m) of the NEA and plays a key role in the determination of circulation patterns and the generation of various frontal and eddy structures. Here we are concerned with the examination of mesozooplankton assemblages in the NEA during the thermal stratification period (July 2004) in two discrete sampling layers: (a) Layer 1 (from the base of halocline to the surface: ~0–20 m) which is directly influenced by BSW; and (b) the deeper ~20–50 m layer (Layer 2). Our main objective was to assess the response of mesozooplankton to the BSW and the associated hydrological structures. In July 2004, the BSW was mainly restricted in the eastern part of the NEA where it was entrapped in a ~50-km wide anticyclonic gyre (Samothraki gyre). A marked spatial differentiation in mesozooplankton assemblage structure, significantly related to this hydrodynamic partitioning, was detected in Layer 1. Sampling sites under the direct influence of low salinity–high temperature gyre waters were characterized by a considerably higher mesozooplankton stock than the remaining area, mainly due to the outstanding numerical dominance of the cladoceran species Penilia avirostris. Copepods displayed notably low densities within the gyre and low species diversity, the calanoid Temora stylifera was the only abundant species. The mesozooplankton community outside the gyre zone shifted towards lower levels of total abundance, with a lesser contribution of cladocerans and an increase in the importance of small-sized copepods (e.g. Acartia clausi, Paracalanus parvus, copepodites of Oithona spp. and Clausocalanus spp.). In the subsurface layer (Layer 2), the mesozooplankton community also exhibited spatial heterogeneity which could be hardly explained by variability in environmental parameters. The periphery of the anticyclone below the halocline was distinguished from the remaining neritic area, presenting markedly high mesozooplankton productivity and distinct group composition. An inverse pattern in the mesozooplankton stock vertical distribution was observed at the periphery of the gyre (Layer 2> Layer 1) comparative to the remaining sites (Layer 1 > Layer 2), which was mainly due to unusually high concentrations of surface-living zooplankters below the halocline. The latter could be explained in terms of expected water flow patterns in an anticyclonic eddy.