It is generally believed that maize (Zea mays L. ssp. mays) arose as a tetraploid; however, the two progenitor genomescannot be unequivocally traced within the genome of modern maize. We have taken a new approach to investigatethe origin of the maize genome. We isolated and sequenced large genomic fragments from the regions surroundingfive duplicated loci from the maize genome and their orthologous loci in sorghum, and then we compared thesesequences with the orthologous regions in the rice genome. Within the studied segments, we identified 11 genes thatwere conserved in location, order, and orientation. We performed phylogenetic and distance analyses and examinedthe patterns of estimated times of divergence for sorghum and maize gene orthologs and also the time of divergencefor maize orthologs. Our results support a tetraploid origin of maize. This analysis also indicates contemporaneousdivergence of the ancestral sorghum genome and the two maize progenitor genomes about 11.9 million years ago(Mya). On the basis of a putative conversion event detected for one of the genes, tetraploidization must haveoccurred before 4.8 Mya, and therefore, preceded the major maize genome expansion by gene amplification andretrotransposition.