In Saccharomyces cerevisiae, ethyl caprylate is produced by the esterification of caprylic acid, which is synthesized through the action of fatty acid synthase. A recent study reported a yeast mutant with a single nucleotide substitution in the alpha subunit of fatty acid synthase (FAS2) gene (F1279Y; 3836T>A) that produced large amounts of ethyl caprylate. Here, we designed two primer sets (P1/P2 and P3/P4) with mismatches that incorporate restriction sites for the enzymes NdeI and SspI, respectively and developed an easy and rapid polymerase chain reaction-restriction fragment length polymorphism assay to identify yeasts harboring the FAS2-F1279Y mutation associated with high ethyl caprylate productivity.