The identification of differentially expressed genes has important implications in understanding the biology of colorectal tumorigenesis and progression, as well as developing new diagnostic and therapeutic strategies. In this study, cDNA microarray technology was used to identify colorectal tumor-related functional genes, which are overexpressed continuously from colorectal adenoma to adenocarcinoma. A set of 23 genes with progressive overexpression in the development of colorectal cancer (CRC) was identified by cDNA microarray, then analyzed by sequencing and Northern blot analysis. Validation of our array results was simultaneously performed by exploring the SAGEmap database. Furthermore, the gradually over-expressed genes from adenoma to adenocarcinoma were validated by Northern blot analysis with additional samples from three patients with synchronous colorectal adenocarcinoma and adenoma and four patients with CRC. Of these 23 genes, one was a function-unknown gene, designated as Homo sapiens chromosome 21q22.1 anonymous mRNA sequence (Genbank accession no. AF003738), and 22 were function-known genes. Searching through the Gene Ontology Browser at the Cancer Genome Analysis Project website revealed that the biological roles of these 22 function-known genes are involved in cell motility, cell adhesion, chemokine activity, signal transduction, cytoskeleton organization, proteolysis, apoptosis, and cell proliferation. The genes identified in the present study offer valuable information on colorectal carcinogenesis and metastasis, and represent a potential source of novel targets for new strategies for CRC diagnosis and therapy.