Polyploidy is viewed as an important mechanism of sympatric speciation, but few studies have documented the reproductive barriers between polyploids and their diploid progenitors or explored the significance of assortative mating for polyploid establishment. Here we synthesize new and existing data on five prezygotic (geographic isolation, flowering asynchrony, pollinator fidelity, self-pollination, gametic selection) and two postzygotic (selection against triploid hybrids, inbreeding depression) reproductive barriers between diploid and autotetraploid individuals of the perennial plant Chamerion angustifolium. We also present estimates of realized rates of between-ploidy mating and examine the impact of assortative mating on polyploid dynamics using computer simulation. Reproductive isolation (measured from 0 to 1) was enforced by each barrier, including: geographic separation (RI = 0.41), flowering asynchrony (0.13), pollinator fidelity (0.85), self-pollination (0.44), gametic selection (0.44) and postzygotic isolation (0.87). Total reproductive isolation was 0.997, with the largest relative contributions by geography (41%) and pollinator fidelity (44%). Prezygotic barriers accounted for 97.6% isolation overall; however, tetraploids were more assortatively mating (98%) than diploids (79%). Realized reproductive isolation between ploidy levels in sympatric populations was 87% and tetraploids produced significantly fewer triploids than did diploids. Simulations indicated that the observed prezygotic isolation will reduce the strength of minority disadvantage acting on tetraploids and increase the importance of differences in viability and fertility between cytotypes in regulating polyploidy establishment.