Tuliposides (Pos) are major defensive secondary metabolites in tulip (genus Tulipa), having 4-hydroxy-2-methylenebutanoyl and/or (3S)-3,4-dihydroxy-2-methylenebutanoyl groups at the C-1 and/or C-6 positions of D-glucose. The acyl group at the C-6 position is converted to antimicrobial lactones, tulipalins, by tuliposide-converting enzymes (TCEs). In the course of a survey of tulip tissue extracts to identify novel Pos, we found a minute high-performance liquid chromatography peak that disappeared following the action of a TCE, and whose retention time differed from those of known Pos. Spectroscopic analyses of the purified compound, as well as its enzymatic degradation products, revealed its structure as 5 ''-O-(6-O-(4'-hydroxy-2'-methylenebutanoyl))-beta-D-glucopyranosyl-(2 '' R)-2 ''-hydroxymethyl-4 ''-butyrolactone, which is a novel glucoside ester-type Pos. We gave this compound the trivial name 'tuliposide G' (PosG). PosG accumulated in bulbs, at markedly lower levels than 6-PosA (the major Pos in bulbs), but was not found in any other tissues. Quantification of PosG in bulbs of 52 types of tulip, including 30 cultivars (Tulipa gesneriana) and 22 wild Tulipa spp., resulted in the detection of PosG in 28 cultivars, while PosG was present only in three wild species belonging to the subgenus Tulipa, the same subgenus to which tulip cultivars belong, suggesting the potential usefulness of PosG as a chemotaxonomic marker in tulip.