Thraustochydrids has been known for their ubiquitous distribution in the ocean. However, a few efforts have been made to investigate their ecology. In this study, we have applied molecular method, acriflavine direct detection, and classical oceanographic methods to investigate the abundance and diversity of thraustochytrids in the North Pacific subtropical gyre. Our results revealed interesting temporal and spatial variations of their population. Out of three seasons (spring, summer, and fall), cruise Hawaii Ocean Time-series (HOT)-216 during November 2009 obtained the highest abundance of thraustochytrids ranging from 1,890 (Station S1C1, 45 m) to 630,000 (Station S2C12, 100 m) cells L(-1) of seawater, which accounted for a 0.79 to 281.0 % biomass ratio to that of bacteria in terms of gram carbon per liter. A patchy distribution of these organisms was widely observed in the water column and they were somehow related to the maximum chlorophyll layers. A total of 25 operational taxonomic units (OTUs) from cruise HOT-216 formed four phylogroups in the specific labyrinthulomycetes 18S rRNA-based phylogenetic tree, with the largest group of 20 OTUs fell into the Aplanochytrium cluster and the others aligned with uncultured clones or none, thus appeared to be undescribed. This study indicates the presence of new thraustochytrids lineages and their quantitative importance in the marine water column.