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A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticumaestivum, Agropyronelongatum, Secalecereale, Hordeumvulgare, Alliumcepa, Muscariarmenaticum and Liliumlongiflorum) and Dicotyledonae (Viciafaba, Betavulgaris, Arabidopsisthaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.