Marine heterotrophic microbes are capable of accumulating large amounts of lipids, omega-3 fatty acids, carotenoids, and have potential for biodiesel production. Pollen baiting using Pinus radiata pollen grain along with direct plating techniques were used in this study as techniques for the isolation of oil-producing marine thraustochytrid species from Queenscliff, Victoria, Australia. Thirteen isolates were obtained using either direct plating or using pine pollen, with pine pollen acting as a specific substrate for the surface attachment of thraustochytrids. The isolates obtained from the pollen baiting technique showed a wide range of docosahexaenoic acid (DHA) accumulation, from 11 to 41 % of total fatty acid content (TFA). Direct plating isolates showed a moderate range of DHA accumulation, from 19 to 25 % of TFA. Seven isolates were identified on the basis of 18S rRNA sequencing technique as Thraustochytrium species, Schizochytrium species, and Ulkenia species. Although both methods appear to result in the isolation of similar strains, pollen baiting proved to be a simpler method for the isolation of these relatively slow-growing organisms.