QTLs associated with products of the carotenoid pathway, including lycopene and the provitamin A carotenes alpha- and beta-carotene, were investigated in two unrelated F(2) carrot populations, derived from crosses between orange cultivated B493 and white wild QAL (Population 1), and orange cultivated Brasilia and dark-orange cultivated HCM (Population 2). The mapping populations of 160 and 180 individuals, respectively, were analyzed with single-marker and interval-mapping statistical approaches, using coupling linkage maps for each parent. Single markers were selected for further analysis based on the Wilcoxon sum-rank non-parametric test. Interval mapping performed with Population 2 detected four, eight, three, one and five putative QTLs associated with accumulation of xi-carotene, alpha-carotene, beta-carotene, lycopene and phytoene, respectively. Among these, the major QTLs explained 13.0%, 10.2%, 13.0%, 7.2% and 10.2% of total phenotypic variation. In Population 1 single-marker analysis identified loci explaining up to 13.8%, 6.8%, 19.3%, 5.7%, and 17.5%, respectively, of total phenotypic variation for these same carotenoids. Overall analysis demonstrated clustering of these QTLs associated with the carotenoid pathway: the AFLP loci AACCAT178-Q and AAGCAG233-Q, on linkage group 5, explained 17.8%, 22.8% and 23.5% of total phenotypic variation for zeta-carotene, phytoene and beta-carotene in Population 1. Two major clusters of QTLs, with LOD scores greater than 1.8, mapped to intervals no larger than 2 cM for zeta-carotene, beta-carotene, alpha-carotene and lycopene on linkage group 3, and for zeta-carotene and phytoene on linkage group 9, and these explained 3.7% to 13.0% of variation for each carotenoid product. Thus, these results suggest that clustering of related pathway loci is favored during evolution, since closely linked "pathway mates" are not easily separated by recombination.