After the success of comparative genomics studies, comparative transcriptomics have gained in importance. A number of methodological drawbacks, however, prevent us from fully understanding expression evolution: target species are limited to very closely related species, and the number of target genes is small. In this study, we propose a new microarray to measure multi-species gene expression levels, in which orthologous genes are measured with a single probe. We checked the effects of probe sequence mutations on expression levels using systematically mutated probes, and found that mutations on the 3'-end of probes have very little effect on expression values whereas those on the 5'-end decreased the observed expression levels. According to the results, we generated an array for humans, rats and mice that covered 6683 genes. Both the species-range and the number of genes are larger than those of previous arrays.