Polyploid evolution is often considered a mechanism of instant speciation; yet the establishment of rare tetraploids within diploid populations may be constrained by a frequency-dependent mating disadvantage (minority cytotype exclusion principle). I tested this hypothesis using experimental populations of Chamerion angustifolium (Onagraceae) that contained di¡erent proportions of tetraploids and diploids. Fitness, measured as total seed production over the entire £owering season, was calculated from a census of £ower number and estimates of ovule number per £ower and proportion of seed set per fruit. The ¢tness of tetraploids relative to diploids was frequency dependent, increasing from 0.4, when tetraploids were rare, to 0.7 when at 50% and 1.15 when they were in the majority (67%). This pattern exists because of a negative relationship between tetraploid frequency and seed set per fruit in diploids. Seed set in tetraploids was independent of cytotype frequency. The frequency-independent e¡ect in tetraploids re£ects higher assortative mating, partly because of non-random patterns of bee visitation. Bees visited a disproportionately high number of diploid in£orescences; however, the proportion of successive £ights between tetraploids increased above random expectations as the frequency of tetraploids decreased. These results provide the ¢rst experimental test of frequency-dependent ¢tness in diploid^polyploid mixtures and suggest an important role for more gradual, population processes governing the evolution of polyploidy in natural populations.