Alliinase operates in the biochemical pathway that produces the compounds responsible for the characteristic flavor of onion. We isolated and characterized the 86-kb BAC clone containing a novel onion alliinase gene, ALL1. Identity of deduced amino acid sequence of ALL1 with a bulb alliinase is 65.4% and with a root alliinase is 67.3%. The ALL1 gene is expressed specifically in onion roots and estimated pI value of mature ALL1 protein is similar to that of root alliinase isoform I, which is an uncharacterized protein having alliinase activity. The highly repetitive sequences around the ALL1 gene was observed from sequence and DNA gel blot analyses. The 33.2% G+C content of the 35-kb ALL1 region is similar to that of dicot plants and lower than that of monocot cereal plants, although onion is classified into monocots. The present study shows the first evidence of the onion genomic sequences around genes differed from the cereal genome.