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Many times small differences in genome size are reported between or within plant species in which no cytologic confirmation is made. Attempts to repeat these studies have met with limited success. The controversy then becomes whether or not these small differences that were not confirmed cytologically are real. The present study was undertaken to determine if the ≈1% nuclear variation seen by flow cytometry among wheat lines selected for aluminum response was due to actual chromatin differences. The three parental wheat cultivars used in the aluminum selection along with the isolines resulting from the selection were analyzed. One parental line had previously been reported to have alien chromatin substituted for the corresponding wheat homoeologous chromatin. Genomic in situ hybridization was used to determine the presence or absence of rye chromatin in three cultivars and six near-isolines of wheat. Upon observing metaphase chromosomes of the Century parent and its isolines, two of the chromosomes were observed to be one-half yellow-orange, indicating rye chromatin with the remaining portion of the chromosomes and the other 40 wheat chromosomes having no label indicating wheat chromatin. In the Chisholm parent and its isolines, none of the chromosomes were labeled, indicating the absence of rye chromatin. In addition, none of the third parents' chromosomes had the rye yellow-orange signal. The wheat lines with the larger DNA contents were observed to have alien DNA present. DNA differences between the normal wheat chromosomes and the substituting alien chromatin were calculated based on total chromosome length. The increase in genome size of the wheat lines containing the alien chromatin appears to be the result of the alien chromatin having ≈43% more DNA than the wheat chromatin it is replacing. Thus, the small DNA difference previously reported by flow cytometry was demonstrated to be a real DNA variation due to the presence of small fragments of alien chromosomes added to the wheat genome. Cytometry 41:36–40, 2000 © 2000 Wiley-Liss, Inc.