Acylsugars exuded from type IV trichomes mediate the multiple pest resistance found in the wild tomato species, Lycopersicon pennellii. A marker-assisted selection breeding program was used to attempt the transfer of the ability to accumulate acylsugars to cultivated tomato. RFLP and PCR-based markers were used through three backcross generations to select plants containing 5 target regions associated by QTL analysis with acylsugar accumulation. The BC1F1 plant selected possessed all 5 target regions and accumulated acylsugars at a moderate level similar to that of the interspecific F-1 control. The BC2F1 and BC3F1 selections contained complementary subsets of the 5 target regions and did nor accumulate acylsugars. BC3F1 plants with complementary subsets of the 5 target regions were intermated to produce populations segregating for the 5 target regions. From 1000 BC3F1-intermated plants, three plants were found which accumulated acylsugars at low levels and contained 3 to 5 of the target regions. The recovery of acylsugar accumulation in progeny of the intermated BC3F1 plants supports the involvement of at least some of the 5 target regions in acylsugar biosynthesis. However, since the levels of acylsugars accumulated by these plants were lower than that of the interspecific F-1, it is likely that another, as of yet unidentified, region is necessary for accumulation of higher levels of acylsugars.