A central problem in speciation is the origin and mechanisms of reproductive barriers that block gene flow between sympatric populations. Wind-pollinated plant species that flower in synchrony with one another rely on post-pollination interactions to maintain reproductive isolation. In some locations in Mexico, sympatric populations of domesticated maize and annual teosinte grow in intimate associate and flower synchronously, but rarely produce hybrids. This trait is typically conferred by a single haplotype, Teosinte crossing barrier1-s. Here, we show that the Teosinte crossing barrier1-s haplotype contains a pistil-expressed, potential speciation gene, encoding a pectin methylesterase homolog. The modification of the pollen tube cell wall by the pistil, then, is likely a key mechanism for pollen rejection in Zea and may represent a general mechanism for reproductive isolation in grasses.