Information on genetic diversity of picoeukaryotes (<2 to 3 µm) comes mainly from traditional gene cloning and sequencing. However, the method suffers from cloning biases and limited throughput. We studied the composition and genetic diversity of picoeukaryotes off the subtropical western Pacific coast using cloning-independent and massively parallel 454 pyrosequencing of the hypervariable V4 region of the 18S rRNA gene. The approach gave a high coverage of the community at genetic difference >=5% but still underestimated the total diversity at genetic difference <=2%. Picoeukaryotic assemblage in the eutrophic site was less diverse than that with low chlorophyll a biomass. Stramenopiles, dinoflagellates, ciliates and prasinophytes were the dominant groups, comprising approximately 29, 19, 11 and 11% of the picoeukaryotes respectively. A differential spatial distribution of high-level taxonomic groups and phylotype OTUs of picoeukaryotes was observed between samples. Our study represents the most comprehensive examination of marine picoeukaryotic diversity to date, using the 454 sequencing-by-synthesis technology for the first time.