Lycopene content is a key component of tomato (Solanum lycopersicum L.) fruit quality, and is a focus of many tomato-breeding programs. Two QTLs for increased fruit lycopene content, inherited from a high-lycopene S. pimpinellifolium accession, were previously detected on tomato chromosomes 7 and 12 using a S. lycopersicum × S. pimpinellifolium RIL population, and were identified as potential targets for marker-assisted selection and positional cloning. To validate the phenotypic effect of these two QTLs, a BC2 population was developed from a cross between a select RIL and the S. lycopersicum recurrent parent. The BC2 population was field-grown and evaluated for fruit lycopene content using HPLC. Statistical analyses revealed that while lyc7.1 did not significantly increase lycopene content in the heterozygous condition, individuals harboring lyc12.1 in the heterozygous condition contained 70.3 % higher lycopene than the recurrent parent. To eliminate the potential pleiotropic effect of fruit size and minimize the physical size of the lyc12.1 introgression, a marker-assisted backcross program was undertaken and produced a BC3S1 NIL population (n = 1,500) segregating for lyc12.1. Lycopene contents from lyc12.1 homozygous and heterozygous recombinants in this population were measured and lyc12.1 was localized to a 1.5 cM region. Furthermore, we determined that lyc12.1 was delimited to a ~1.5 Mb sequence of tomato chromosome 12, and provided some insight into potential candidate genes in the region. The derived sub-NILs will be useful for transferring of lyc12.1 to other tomato genetic backgrounds and for further fine-mapping and cloning of the QTL.