This study describes the high-fidelity DNA histograms in different stages of neoplastic progression to Barrett's adenocarcinoma (BAC). High-fidelity DNA histograms were obtained with image cytometry on sections, and were classified based on DNA index values of the peaks into diploid, mild aneuploid, moderate aneuploid and severe aneuploid. Heterogeneity index (HI) representing cells with different DNA content and the 5N exceeding cell fraction were determined. One hundred and eighty-seven cases, including 34 normal gastrointestinal mucosa (control), 66 Barrett's-specialized intestinal metaplasia (SIM), 22 low-grade dysplasia (LGD), 22 high-grade dysplasia (HGD) and 43 BAC were investigated. Controls showed sharp diploid peaks with HI values less than 13, and no 5N exceeding nuclei. SIM showed a spectrum of histograms including diploid, mild aneuploid and moderate aneuploid histograms. The frequency and severity of aneuploidy increased with worsening histological grades of dysplasia. All BAC cases were aneuploid, with moderate or severe aneuploidy. Marked elevated HI values (>20) and 5N exceeding fractions (>5%) were found in 5%, 32%, 50% and 88% of cases with SIM, LGD, HGD and BAC, respectively. The high-fidelity DNA histograms suggest that (1) Barrett's SIM may already be dysplastic in nature, and all BAC may be markedly aneuploid; and (2) elevated cellular DNA heterogeneity and 5N fractions may be markers of progressive chromosomal changes and ‘unstable aneuploidy’ that identifies progressive lesions.