We report the first isolation and characterization of insect fatty acid Δ12-desaturase genes, AdD12Des from house cricket (Acheta domesticus) and TcD12Des from the red flour beetle (Tribolium castaneum), responsible for the production of linoleic acid from oleic acid. Sequence analysis shows the cricket and flour beetle Δ12-desaturase genes have evolved independently from all previously known Δ12-desaturases and are much more closely related to the archetypal stearoyl-Coenzyme A-acting desaturase from rat than to the phospholipid-acting Δ12-desaturases widely reported in plants. Phylogenetic and functional analysis indicates the cricket AdD12Des gene may have evolved from an ancestral Δ9-desaturase. By contrast, the beetle Δ12-desaturase is distantly related to the cricket genes and beetle Δ9-desaturases suggesting evolution by an independent route. Linoleic acid has key physiological roles in insects and this is the first report of genes capable of producing this essential fatty acid in higher animals.