KCS (3-ketoacyl-CoA synthase) is the key enzyme catalyzing the first step of very long chain fatty acid (VLCFA) biosynthesis. Studies showed that different KCSs possessed different substrate preference. Malania oleifera are abundance of VLCFAs in its mature seeds, especially the nervonic acid, which is essential for human health. In this study, we identified and characterized 18 KCS genes in M. oleifera genome. Phylogenetic analysis showed that these KCS genes were classified into four subfamilies, including two FAE-like, six KCS-like, eight FDH-like and two CER6. We concentrated on the functional role of two FAE-like genes, Maole003085.T1 and Maole004215.T1 which encoded predicted amino acid residues of 516 and 518 in protein, respectively. Multiple sequence alignment showed that their two proteins contained the known and conserved active sites among FAE-like subfamily. Upon heterologous expression in wild type yeast (Saccharomyces cerevisiae) INVSc1, we found that Maole004215.T1 could produce four new fatty acids including C22:0/C22:1 and C24:0/C24:1, but Maole003085.T1 only produced C22:1. Besides, upon heterologous expression in mutant yeast BY4741-△elo3, we found the Maole003085.T1 could produce C24:0 and C26:0, while the Maole004215.T1 could catalyze the formation of fatty acids C24:0, C26:0 and C28:0. These results showed Maole003085.T1 and Maole004215.T1 had fatty acid elongation activity in yeast, and possessed different substrate preference in the production of different VLCFAs. Interestingly, we found Maole004215.T1 could produce nervonic acid in yeast, which provides molecular basis on the genetic improvement and genic engineering for producing nervonic acid resources by using biotechnological methods.