We report a global gene for gene alignment of the genomes of Brassica oleracea and Arabidopsis thaliana by construction of a transcriptome map based on B. oleracea cDNAs obtained from leaf tissue. cDNAs were synthesized from total RNA extracted from individual F2s of a mapping population resulting from crossing double-haploids of broccoli and cauliflower. The map consisted of 247 cDNA markers obtained by the SRAP technique. After sequencing 190 of the polymorphic cDNA bands, FASTA detected 169 sequences with similarity to genes reported in Arabidopsis. There was extensive colinearity between the two genomes for chromosomal segments rather than for whole chromosomes, often showing inversions and deletions/insertions. Large-scale duplications were observed in the B. oleracea genome, but were unevenly distributed, arguing against ancient triplication of the entire genome. The most duplicated segments corresponded to those found on Arabidopsis chromosomes 1 and 5, whereas chromosomes 2 and 4 were the least represented in Brassica. Clear differences in the similarity score value of related sequences allowed the identification of orthologs. Transcriptome mapping is an efficient approach that allows gene-for-gene alignment between a fully sequenced and a poorly characterized genome.