Industrial oil flax (Linum usitatissimum) and edible oil or solin flax differ markedly in seed linolenic acid levels. Despite the economic importance of low-linolenic-acid or solin flax, the molecular mechanism underlying this trait has not been established. Two independently inherited genes control the low-linolenic-acid trait in flax. Here, we identified two genes, LuFAD3A and LuFAD3B that encode microsomal desaturases capable of desaturating linoleic acid. The deduced proteins encoded by these genes shared 95.4% identity. In the low-linolenic-acid line solin 593-708, both LuFAD3A and LuFAD3B carry point mutations that produce premature stop codons. Expression of these genes in yeast (Saccharomyces cerevisiae) demonstrated that, while the wild-type proteins were capable of desaturating linoleic acid, the truncated proteins were inactive. Furthermore, the low-linolenic-acid phenotype in flax was complemented by transformation with a wild-type gene. Codominant DNA markers were developed to distinguish between null and wild-type alleles of both genes, and linolenic acid levels cosegregated with genotypes, providing further proof that LuFAD3A and LuFAD3B are the major genes controlling linolenic acid levels in flax. The level of LuFAD3 transcripts in seeds peaked at about 20 d after flowering, and transcripts were not detectable in leaf, root, or stem tissue. A dramatic reduction in transcript levels of both genes occurred in the low-linolenic-acid solin line, which was likely due to nonsense-mediated decay.