2,4,6-Trichloroanisole (TCA), which has been identified as the main component responsible for the cork taint in wine, was detected in sake samples having a musty/muddy off-flavor by stir bar sorptive extraction (SBSE). We confirmed that TCA is one of the components causing this off-flavor in sake, as in other alcoholic beverages, from a sensory analysis showing the correlation between TCA concentration and the intensity of the musty/muddy off-flavor. We investigated the route of TCA production in the rice koji preparation process and in the moromi mash process for sake brewing. We found that TCA is produced mainly by the biomethylation of 2,4,6-trichlorophenol (TCP) by rice koji in brewing and that TCP originates from the wooden tools used in preparing rice koji.