Adenocarcinoma in Barrett's esophagus is the second most rapidly increasing cancer in western society. The cause and pathogenesis are unknown. Although histological studies suggest that there is successive progression from metaplasia and dysplasia, with a high risk of subsequent invasive carcinoma, at present there is no direct evidence that metaplastic and dysplastic epithelia are clonal precursors of adenocarcinoma. We selected 12 esophagectomy specimens of Barrett's esophagus patients, which showed a spectrum of normal tissue, metaplasia, dysplasia, and invasive carcinoma in each individual biopsy. We applied the microdissection technique to selectively procure microscopic tissue samples from H&E-stained slides for genetic evaluation using polymorphic markers flanking the APC gene locus. Identical APC gene alterations were found in the dysplastic and adenocarcinoma foci of all informative cases. The same changes were observed even in some metaplastic foci adjacent to dysplasia. Furthermore, clonality analysis of X-chromosome inactivation in female cases verified the same X-chromosome inactivation pattern in carcinoma, dysplasia, and metaplasia adjacent to dysplasia. No APC gene alterations were found in the normal epithelium and metaplasia distant from dysplasia. These data show for the first time that a tissue field of genotypic changes precedes the histopathological phenotypic changes of carcinoma in Barrett's esophagus syndrome. Our findings, in conjunction with the applied tissue microdissection technique, may help identify genotypic changes in patients with Barrett's esophagus before phenotypic changes occur. Therefore, genotyping of Barrett's metaplastic epithelium may supplement the histopathological evaluation of Barrett's esophagus.