To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, β-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low- and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of β-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high- and low- risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.