The species composition of plankton is essential to understand the material and energy cycling within marine ecosystem. It also provides the useful information for understanding the properties of marine environments due to its sensitivity to the physicochemical characteristics and variability of water masses. In this study we adopted metagenomics to evaluate eukaryotic plankton species diversity from coastal waters off Busan. Characteristics of water masses at sampling sites is expected to be very complex due to the mixing of various water masses; Nakdong River runoff, Changjiang diluted water (CDW), South Sea coastal water, and Tsushima warm current. 18S rDNA clone libraries were constructed from surface waters at the three sites off Busan. Clone libraries revealed 94 unique phylotypes from 370 clones; Dinophyceae(42 phylotypes), Ciliophora(15 phylotypes), Bacillariophyta(7 phylotypes), Chlorophyta(2 phylotypes), Haptophyceae(1 phylotype), Metazoa(Arthropoda( 17 phylotypes), Chaetognatha(1 phylotypes), Cnidaria(2 phylotypes), Chordata(1 phylotype)), Rhizaria (Acantharea(2 phylotypes), Polycystinea(1 phylotype)), Telonemida(1 phylotype), Fungi(2 phylotypes). The difference in species diversity at the closely located three sites off Busan may be attributed to the various physicochemical properties of water masses at these sites by the mixture of water masses of various origins. Metagenomic study of species composition may provide useful information for understanding marine ecosystem of coastal waters with various physicochemical properties in the near feature.