Marine algae are a major source of omega-3/6-long-chain polyunsaturated fatty acids (ω3/6-LCPUFAs), which are essential nutrients. In marine algae, the biosynthesis of these molecules begins with the desaturation of fatty acids by membrane bound Δ6 desaturases. In the absence of a molecular structure, the structural basis for the ω3/6-specificity of Δ6 desaturases is poorly understood. In order to gain more insight into the substrate specificity of these enzymes, which can discriminate their substrates from structurally-similar compounds that differ by only a single double-bond, we have conducted a consensus mutagenesis and ancestral reconstruction-based analysis of the Δ6 desaturase family, focusing on the Δ6 desaturase from Micromonas pusilla (MpΔ6des). Point mutants of MpΔ6des were constructed based on consensus mutagenesis analyses from three different sets of related desaturases and analysed in terms of their expression and activity. Analysis of the variants highlights the importance of the residues near the putative active site in determining substrate preference. Surprisingly, the N-terminal cytochrome b5 domain, previously thought to act only in electron transfer during catalysis, was also a major contributor to substrate specificity. Further sequence-based analysis of the Δ6 desaturase family allows us to propose a mechanism by which this domain might be implicated in enzyme specificity. Ancestral reconstruction also suggests that some extant Δ6 desaturases have evolved to become generalists from an ω3- specific ancestor in response to environmental changes. This dataset provides a detailed map of regions within enzymes of this family that contribute to substrate specificity, as well as allowing us to propose a potential mechanism for the contribution of the b5 cytochrome domain to the incredible substrate specificity of these enzymes. This, in turn, should facilitate future attempts to engineer and tailor the catalytic specificity of this important enzyme family.