The evolution of genomes can be studied by comparing maps of homologous genes which show changes in nucleic acid sequences and chromosome rearrangements. In this study, we developed a set of 32 amplified consensus gene markers (ACGMs) that amplified gene sequences from Arabidopsis thaliana and Brassica napus. Our methodology, based on PCR, facilitated the rapid sequencing of homologous genes from various species of the same phylogenetic family and the detection of intragenic polymorphism. We found that such polymorphism principally concerned intron sequences and we used it to attribute a Brassica oleracea or Brassica rapa origin to the B. napus sequences and to map 43 rapeseed genes. We confirm that the genetic position of homologous genes varied between B. napus and A. thaliana. ACGMs are a useful tool for genome evolution studies and for the further development of single nucleotide polymorphism suitable for use in genetic mapping and genetic diversity analyses.