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Phytoplankton are primary producers and can be important indicators of environmental change. To monitor the plankton species composition of environmental seawater samples, we developed a molecular method composed of colony polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and sequencing. A clone library of the ribosomal small subunit RNA gene (18S rDNA) in the nuclear genome was constructed by environmental PCR using a newly designed primer set and clones were directly amplified by colony PCR. To select unique putative clones, we choose a PCR-RFLP method that employed two restriction enzymes ( MseI and Tsp509I). After the PCR-RFLP pattern was evaluated, selected clones were sequenced and analyzed. In this study, we revealed the hidden biodiversity in environmental seawater containing a wide range of taxonomic groups in the Alveolata (Ciliphora and Dinophyceae), Euglenozoa, Stramenopiles (Bacillariophyta), and Viridiplantae (Chlorophyta) without the need to conduct extensive colony isolation techniques. Moreover, we found species of fungi and Metazoa (Arthropoda, Annelida, and Mollusca). Therefore, this improved molecular method can be used to generate a robust database describing the species diversity of environmental samples and provide useful information regarding the dynamics of the eukaryotic plankton community structure. [ABSTRACT FROM AUTHOR]