Single chromosome identification is of primal importance in the study of evolutive process in complex genomes, such as polyploidization and hybridization events, among others. Traditionally, chromosome identification is made by arranging the chromosomes by the length of the short (p) and the long (q) arms, identifying the centromeres and secondary constrictions. Furthermore, accurate chromosome identification can be performed trough chromosome differentiation techniques such as C-, N-, and Q-banding. In the last years, through the development of fluorescent in situ hybridization techniques (FISH), even more accurate single chromosome identification has been accomplished, by the hybridization of highly conserved repetitive sequences, such as rDNAs. However, particular probes must be developed through laborious isolation and cloning molecular techniques. The comparative genomic in situ hybridization (cGISH) is a straightforward technique that allows the identification of single chromosomes by the generation of signals of conserved DNA regions along the chromosomes of different species. In this study we labeled total genomic DNA of Triticum aestivum and Arabidopsis thaliana and hybridized it to chromosomes of different cultivars of the genus Lilium. Different stringencies were applied to determine the optimum removal of cross hybridization, the 80% stringency showed to be the best, giving a clear signal and removing most of the cross hybridization. Triticum aestivum total genomic DNA exhibited six landmarks on three homologous chromosomes in the three different cultivars while Arabidopsis thaliana total genomic DNA exhibited six landmarks on three homologous chromosomes of Lilium, one of these signals being in a different chromosome of those of the T. aestivum signals. Together with the DAPI bands the total genomic DNAs landmarks allowed the identification of six out of 12 single homologous chromosomes
