Malic enzyme (ME) is one of the important enzymes for furnishing the cofactor NAD(P)H for the biosynthesis of fatty acids and sterols. Due to the existence of multiple ME isoforms in a range of oleaginous microbes, a molecular basis for the evolutionary relationships amongst the enzymes in oleaginous fungi was investigated using sequence analysis and structural modelling. Evolutionary distance and structural characteristics were used to discriminate the MEs of yeasts and fungi into several groups. Interestingly, the NADP + -dependent MEs of Mucoromycotina had an unusual insertion region (FLxxPG) that was not found in other fungi. However, the subcellular compartment of the Mucoromycotina enzyme could not be clearly identified by an analysis of signal peptide sequences. A constructed structural model of the ME of Mucor circinelloides suggested that the insertion region is located at the N-terminus of the enzyme (aa 159-163). In addition, it is presumably part of the dimer interface region of the enzyme, which might provide a continuously positively charged pocket for the efficient binding of negatively charged effector molecules. The discovery of the unique structure of the Mucoromycotina ME suggests the insertion region could be involved in particular kinetics of this enzyme, which may indicate its involvement in the lipogenesis of industrially important oleaginous microbes.