Members of the genus Lesquerella produce seed oil that contains a high proportion of hydroxy fatty acids (HFAs). There are three groups of Lesquerella species that are distinguished by their most abundant seed oil fatty acid: lesquerolic acid (20:10H; e.g. Lesquerella fendleri), densipolic acid (18:20H; e.g. Lesquerella kathryn), and auricolic acid (20:20H; e.g. Lesquerella auriculata). To investigate the biochemistry of HFA production in Lesquerella species, the conversion of putative radiolabeled intermediates of HFA biosynthesis, including 18:1, 20:1, 18:10H, 18:20H, and 20:10H, was examined in developing embryos of L. fendleri, L. kathryn, and L. auriculata. The results are consistent with (a) 18:10H formation by hydroxylation of 18:1, (b) elongation and desaturation of 18:10H to produce 20:10H and 18:20H, respectively, and (c) desaturation of 20:10H to produce 20:20H. The desaturation of 20:10H was also found to occur in developing embryos of high, but not low, linolenic acid flax. This suggests that the desaturation is catalyzed by the extraplastidial linoreate desaturase. Confirming this suggestion, it was notable that 18:10H and 18:20H were found in low and high linolenic flax (Linum usitatissimum) seeds, respectively, at levels of 0.2 to 1%.