Neoplastic progression is characterized by clonal expansion of tumor cells associated with accumulation of mutational damage. The timing of mutation acquisition could be of value in distinguishing preneoplastic conditions from early and advanced cancer as well as characterizing tumor aggressiveness and treatment response. Using quantitative methods applied to microdissected cell clusters selected according to cytomorphologic features, we sought to demonstrate the feasibility and efficacy for determining the time and course of mutation accumulation in pancreatobiliary cytology specimens. In all, 40 pancreatic duct and 21 biliary brushing cytology specimens were retrieved from the cytology database. Xylene-resistant markings were placed on the slide underside and coverslips removed. Clusters of benign, atypical and malignant cells were manually microdissected and DNA extracted. Mutations (allelic imbalance) (loss of heterozygosity) were quantitatively determined for a broad panel of 15 markers (1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, 22q) as well as point mutation in K-ras-2 using PCR/capillary electrophoresis. Time course was based on earlier mutations having a higher proportion of mutant DNA for a particular marker. The descending frequency of detectable mutational involvement in pancreatic cytology was K-ras-2 point mutation (58%), 3p25-26 and 17q21 (35%), 5q23 (33%), 1p36 (28%), followed by the remaining molecular markers. The descending frequency of mutational content in bile duct cytology was 17p13, 1p36, 3p25-26, and 5q23 followed by remaining molecular markers. K-ras-2 point mutation was not seen in bile duct specimens. While there was overlap in the spectrum of mutational markers in pancreatic duct and biliary brushing cytology, the temporal profile was significantly different (P<0.001). Pancreatic and biliary neoplasia progression involves distinct subset of accumulated defined mutations. Determination of timing of the mutational damage in cytologic material could be incorporated in the work-up and help in making a more definitive diagnosis of malignancy in pancreatobiliary cytology specimens.