omega-3 fatty acid desaturase is a key enzyme for the biosynthesis of omega-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three omega-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new omega-3 desaturases share 55 % identity at the amino acid level with the known Delta-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Delta-12/Delta-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the omega-6 arachidonic acid to the omega-3 eicosapentanoic acid, with a substrate conversion efficiency of 54-65 %. These enzymes have a broad omega-6 fatty acid substrate spectrum, including both C18 and C20 omega-6 fatty acids although they prefer the C20 substrates, and have strong Delta-17 desaturase activity but weaker Delta-15 desaturase activity. Thus, they belong to the Delta-17 desaturase class. Unlike the previously identified bifunctional Delta-12/Delta-15 desaturase from F. monoliforme, they lack Delta-12 desaturase activity. The newly identified Delta-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Delta-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce omega-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.