For identification of ECS (``evolutionarily conserved segments'') between rat and mouse, 893 rat–mouse orthologous gene-pairs were brought together with zoo-FISH analysis. In total, 59 autosomal ECS and 4 X-chromosomal ones were detected. Combining FISH and zoo-FISH data, the segments were anchored on the rat chromosomes, providing an improved comparative map between the two species. Since chromosomal evolution is a slow process, it is reasonable to assume that the genome organization, including gene order, is essentially conserved within the ECS. In this way we assigned tentative subchromosomal map positions to 303 rat genes, for which no regional mapping information was available. Furthermore, the concept of prediction mapping was extended to unmapped rat homologs of genes, which in the mouse are situated inside or in the vicinity of an ECS. For a total of 6669 genes, we predicted a single rat chromosomal position, whereas for another 448 genes we could predict that they were located in one of two possible positions. Thus, our study has increased the number of genes for which there is positional mapping information in the rat almost fivefold.