Consumption of flavonoids found in fruits and vegetables is linked to beneficial health outcomes. Tomato is among the most widely consumed and economically important vegetables worldwide and improvement of the nutrient content could lead to significant health benefits. Rutin, (quercetin-3-rutinoside), the main flavonol in tomato fruit, is confined to the peel. Rutin synthesis is limited by low expression of chalcone isomerase, the enzyme catalyzing the conversion of naringenin chalcone to naringenin quercetin. The wild tomato species Solanum habrochaites is a major source of new alleles to improve cultivated tomato (Solanum lycopersicum). AVRDC—The World Vegetable Center identified introgression line (IL) LA3984 containing a segment of S. habrochaites on chromosome 5 expressing high levels of rutin in full red ripe fruit. An AVRDC high rutin tomato line evaluated for 2 years and two seasons in Taiwan produced mean rutin content about four- to five-fold greater than the mean of all entries and about 11–12 times higher than the commercial fresh market and processing tomato cultivars. The quantitative trait locus (QTL) conditioning high rutin content was mapped to a 0.42 Mb segment on chromosome 5 flanked by markers c2_At3g55120/TaqI and ch05-4.883/ApaLI. Marker c2_Atg55120 overlaps with the chalcone-flavonone isomerase gene Solyc05g10320, and a second chalcone-flavonone isomerase gene is located 3,000 bp upstream from c2_At3g551220. Results of this project will facilitate breeding of high flavonoid tomato lines.