A polymerase chain reaction (PCR)-based approach was employed to isolate putative alleles of the chromoplast-specific lycopene beta-cyclase (CYCB) gene from wild and cultivated tomatoes [Solanum (Section Lycopersicon)]. The objective of this work was to establish an effective PCR protocol by testing DNA samples from distinct germplasm accessions with a primer pair designed to selectively target conserved regions present in the available CYCB sequences. This PCR optimization allowed the amplification of 1219 out 1666 bp of the gene in six taxa: S. cheesmaniae, S. peruvianum, S. neorickii, S. pennellii, S. pimpinellifolium and S. lycopersicum. Sixty-three mutation sites (31 transitions, 18 transversions and 14 single base deletions/insertions) were detected in these accessions when compared to S. lycopersicum AF 254793 (used as reference sequence). The polymorphisms were found predominantly in greenfruited species (20 in S. neorickii, 20 in S. peruvianum, and 32 in S. pennellii). Lower levels of polymorphisms were found in yellow-fruited (three in S. cheesmaniae) and red-fruited species (eight in S. pimpinellifolium and none in the S. lycopersicum). The higher levels of nucleotide diversity in the CYCB-like gene sequences in accessions of greenfruited species as well as the phylogenetic tree agreed with the previous taxonomic studies based upon the granulebound starch synthase gene phylogeny. Sequence analyses of the amplicons obtained via heterologous PCR indicated the CYCB gene-specificity of the primers. Therefore, this PCR-based strategy might be useful to isolate CYCB-like amplicons from other species within the genus Solanum and to develop molecular markers for assisted breeding.