Late blight, caused by Phytophthora infestans deBary, is a major disease of tomato < Lycopersicon esculentum Mill.> in cool, wet environments. In this study we report on the mapping of a gene in L. pimpinellifolium Mill. Accession L3708 conditioning race-specific resistance to P. infestans, F2 tomato plants from the cross CLN657 x L3708 and segregating for late blight resistance were evaluated for resistance to P.infestans isolate Pi-16 that overcomes host resistance genes Ph and Ph-2. Resistant and susceptible DNA pools, created by bulking equal amounts of DNA from nine resistant F 2 plants and eight susceptible F 2 plants , and the DNA pools were used to screen amplified fragment length polymorphic primers, Polymerase chain reaction using 120 combinations of three-base selective nucleotide primer pairs revealed five primer pairs unique to the resistant parent and R-pool. And one pair associated with the susceptible parent and S-pool. Two single copy DNA clones associated with resistance were hybridized with 50 introgression lines derived from L. esculentum xL.pennellii; IL9-3, containing an introgression from the long arm of chromosome 9, was the only polymorphic IL. Restriction fragment length polymorphism markers mapped to chromosome 9 and two AFLP-derived RFLP markers were used to probe a second F 2 population. A major quantitative trait loci with an LOD score of 18.41 was found at RFLP marker TG591;it accounted for 71.4% of the variance. This gene, Ph-3, is non-allelic to tomato late blight resistance genes Ph . Key words Lycopersicon pimpinellifolium Phytophthora infesstans Disease resistance