Studies on autotrophic picoplankton (APP; <3μm) in shallow lakes are mainly confined to the spring through fall seasons, when sampling efforts are not complicated by adverse and unsafe conditions that occur during winter. The aim of the present work was to study the role and diversity of winter APP communities in temperate shallow lakes by means of analysis of measures of size-fractionated photosynthesis and culture-based molecular taxonomic identification. Our results show that APP comprised a substantial part of planktonic primary production in shallow Central European great lakes (13–46% in Lake Balaton and 11–42% in Lake Fertő). Better acclimation of APP than that of the larger phytoplankton (>3μm) to low-temperature and low-light winter environment was confirmed by their higher maximum photosynthetic rate and light utilization parameter. Maximum photosynthetic rate and light saturation parameter increased significantly with both temperature and available light, but with different impact on the two size groups. Twenty-two picoeukaryotic strains were isolated and identified based on 18S rRNA gene sequence analysis. Taxonomic composition of the picoeukaryotic community in the studied shallow lakes was similar to other freshwater lakes in the temperate zone: members of genera Choricystis and Mychonastes were dominant, however, in Lake Balaton, common freshwater taxa such as Stichococcus bacillaris and Nannochloris bacillaris were also found.