cDNA-AFLP is a genome-wide expression analysis technology that does not require any prior knowledge of gene sequences. This PCR-based technique combines a high sensitivity with a high specificity, allowing detection of rarely expressed genes and distinguishing between homologous genes. In this report, we validated quantitative expression data of 110 cDNA-AFLP fragments in yeast with DNA microarrays and GeneChip data. The best correlation was found between cDNA-AFLP and GeneChip data. The cDNA-AFLP data revealed a low number of inconsistent profiles that could be explained by gel artifact, overexposure, or mismatch amplification. In addition, 18 cDNA-AFLP fragments displayed homology to genomic yeast DNA, but could not be linked unambiguously to any known ORF. These fragments were most probably derived from 5′ or 3′ noncoding sequences or might represent previously unidentified ORFs. Genes liable to cross hybridization showed identical results in cDNA-AFLP and GeneChip analysis. Three genes, which were readily detected with cDNA-AFLP, showed no significant expression in GeneChip experiments. We show that cDNA-AFLP is a very good alternative to microarrays and since no preexisting biological or sequence information is required, it is applicable to any species.