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 ?C temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum (C1C1) as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 (G. hirsutum × G. sturtianum) (AtDtC1). 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, At, Dt, and C1, 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 F1 (G. hirsutum × G. australe) (AtAtDtDtG2G2). 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. 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 ° C temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum (C1C1) as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 (G. hirsutum × G. sturtianum) (AtDtC1). Thus, 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 repeated displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, m The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 (G. hirsutum × G. australe) (AtAtDtDtG2G2). 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.