A survey of the three kinetoplastid genome projects revealed the presence of three putative front‐end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Δ8, Δ5 and Δ6 desaturases for L. major, and Δ6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Δ6 desaturases could have Δ4 desaturase activity, based mainly on the conserved HX3HH motif for the second histidine box, when compared with Δ4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front‐end desaturases present in L. major and T. brucei. Our prediction about the presence of Δ4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Δ5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Δ8 desaturase turned out to be a functional Δ6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareΔ6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very‐long‐chain polyunsaturated fatty acid biosynthesis is functional in L. major using Δ6, Δ5 and Δ4 desaturases. In trypanosomes, only Δ4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.