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To investigate differences in culturable freshwater bacterioplankton between summer and winter, we examined specific bacterioplankton in the low temperature surface water of two shallow, hypereutrophic, temperate lakes in Japan over two winters, and compared our results with the previously published data. We used the size-exclusion assay method (SEAM), a cultivation-based approach that simply and effectively isolates typical freshwater bacterioplankton. The specific clusters detected in the winter samples were the Rhodoferax sp. BAL47 cluster (LimA and LimC), a predominant and ubiquitous freshwater lineage, and the LiUU-5-340 cluster. To confirm tolerance to low water temperature of winter-specific groups, we also compared growth rates at 5°C among several pure strains of typical freshwater bacterioplankton clusters belonging to the Rhodoferax sp. BAL47, Polynucleobacter necessarius, GKS98, LiUU-5-340, and IRD18C08 of the class Betaproteobacteria, and the Luna-1 and Luna-2 of the class Actinobacteria. Specific detectability of freshwater bacterioplankton clusters/subclusters by SEAM in the winter sample substantially correlated with the low temperature-specific growth characteristics of each isolate. Response to water temperature is a key control factor in freshwater bacterioplankton assemblage composition. These results provide important insights into the specific response to water temperature of several ubiquitous culturable freshwater bacterioplankton clusters inhabiting a temperate climate zone.