A novel source of resistance to two-spotted spider mite (Tetranychus urticae Koch) was found in Solanum pimpinellifolium L. accession TO-937 and thereby a potential source of desirable traits that could be introduced into new tomato varieties. This resistance was found to be controlled by a major locus modulated by minor loci of unknown location in the genome of this wild tomato. We first applied a bulked segregant analysis (BSA) approach in an F(4) population as a method for rapidly identifying a genomic region of 17 cM on chromosome 2, flanked by two simple sequence repeat markers, harboring Rtu2.1, one of the major QTL involved in the spider mite resistance. A population of 169 recombinant inbred lines was also evaluated for spider mite infestation and a highly saturated genetic map was developed from this population. QTL mapping corroborated that chromosome 2 harbored the Rtu2.1 QTL in the same region that our previous BSA findings pointed out, but an even more robust QTL was found in the telomeric region of this chromosome. This QTL, we termed Rtu2.2, had a LOD score of 15.43 and accounted for more than 30% of the variance of two-spotted spider mite resistance. Several candidate genes involved in trichome formation, synthesis of trichomes exudates and plant defense signaling have been sequenced. However, either the lack of polymorphisms between the parental lines or their map position, away from the QTL, led to their rejection as candidate genes responsible for the two-spotted spider mite resistance. The Rtu2 QTL not only serve as a valuable target for marker-assisted selection of new spider mite-resistant tomato varieties, but also as a starting point for a better understanding of the molecular genetic functions underlying the resistance to this pest.