Lycopersicon pennellii, a wild relative of the tomato, L. esculentum, is resistant to a number of important pests of cultivated tomato due to the accumulation of acylsugars, which constitute 90% of L. pennellii LA716 type IV trichome exudate. An interspecific F2 population created by crossing L. esculentum × L. pennellii was surveyed for acylsugar accumulation and subjected to RFLP analysis to determine the genomic regions associated with the levels of acylglucoses, acylsucroses, and total acylsugars accumulated, and glucose as a percentage of total acylsugars. Data was analyzed using MAPMAKER with and without log10 transformation and using a threshold of either 2.4 (default value for MAPMAKER) or ones calculated according to the Permutation-based Estimated Threshold (PET) method. Genomic regions were identified for each of the traits studied. Effects of analytical method on identification of QTLs, similarities between these results and results published for the genus Solanum, and similarities between these results and the regions transferred by a breeding program selecting for acylsugar production are discussed.