To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization (GISH) technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state: 100:1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 degrees C temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum (C(1)C(1)) as a probe, strong hybridization signals were only observed on chromosomes from the C(1) genome in somatic cells of the hybrid F(1) (G. hirsutum x G. sturtianum) (A(t)D(t)C(1)). Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, A(t), D(t), and C(1), and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F(1) (G. hirsutum x G. australe) (A(t)A(t)D(t)D(t)G(2)G(2)). We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.