Over the past 3 centuries, many species have been dispersed beyond their natural geographic limits by humans, but to our knowledge, reproductive isolation has not been demonstrated for such neo-allopatric species. We grew seeds from three species of Centaurea (Centaurea solstitialis, Centaurea calcitrapa, and Centaurea sulphurea) that are native to Spain and have been introduced into California, and we tested to what extent seed production was affected by pollen source. Compared with within-population crosses, seed production decreased by 52% and 44%, respectively, when C. solstitialis and C. sulphurea from California were pollinated with conspecific pollen from native populations in Spain. This implies rapid evolution of reproductive isolation between populations in their native and nonnative ranges. Whether reproductive isolation has evolved following the introduction of other species is unknown, but additional cases are likely, considering the large number of neo-allopatric species.