Aldehyde dehydrogenase (ALDH) 2 plays a major role in the detoxification of aldehyde and is known to be responsible for alcohol preference. A diminished enzyme activity due to mutation of the Aldh2 gene is associated with high alcohol sensitivity and a low alcohol tolerance in humans. The genomic background distinguishing an alcohol preference and avoidance in various inbred mouse strains is not clear. We created Aldh2-negative mice by transgenic knockout of the Aldh2 gene into the high alcohol preference C57BL/6 background. The Aldh2 gene targeting (Aldh-/-) mice exhibited an alcohol avoidance characteristic. After free-choice ethanol and water drinking, brain and liver acetaldehyde concentrations of Aldh2-/- mice were almost equal to those of wild-type (Aldh2+/+) mice although the Aldh2-/- mice drank less ethanol than the Aldh2+/+ mice. This result indicates that a direct effect of the Aldh2 genotype plays an important role on alcohol preference and acetaldehyde concentration in the brain is correlated with alcohol avoidance. This highlights the potential benefits of alcoholism and alcohol-related disease research in the animal model of ALDH2 alleles.