A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Δ5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain‐of‐function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino‐acid sequence comprised 467 amino‐acid residues containing an N‐terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Δ5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5‐containing Δ5 fatty acid desaturase. As we have already reported one other Δ5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Δ5 fatty acid desaturase genes. The substrate specificities of the two functional Δ5 desaturases of D. discoideum were also examined.