A genomic subtraction between Lactobacillus fructivorans ATCC 8288(T) and an alcoholophilic strain of L. fructivorans (ATCC 15435 strain H-1) was performed. Subtractive DNA fragments were identified to be parts of a 1468-by insertion sequence, a few copies of which were present with in four alcoholophilic strains tested. The insertion sequence, which we named ISLfr1, seems to belong to the ISL3 family. For three alcoholophilic strains, primer walk sequencing revealed that ISLfr1 was inserted into the ORF, which showed homology to accC2 of Lactobacillus plantarum WCFS1. accC2 in one strain seemed to be nonfunctional because of deletion and frameshift mutations, although ISLfr1 was not found in this gene. Although accC2 encodes acetyl-CoA carboxylase, which is essential for lipid metabolism, L. fructivorans was found to have a homolog of accC2. Nonfunctional accC2 might be involved in producing the unusually long acyl chains observed only in alcoholophilic L. fructivorans. From a food engineering standpoint, it appears that the concomitant PCR amplification of ISLfr1 and rDNA is useful for the specific detection of alcoholophilic L. fructivorans.