The first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecific diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSF) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcyb, Lcy-e, Hy-b, and Zep genes labeled with [alpha-P-32]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecular markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the results of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-el, is associated with interspecific diversity in carotenoid accumulation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci