Barriers to interspecific hybridization in Trifolium were investigated by manipulation of mentor pollen treatments, ploidy levels, and compatibility and male sterility systems. Crosses involving the addition of mentor pollen produced fewer seeds and hybrids than crosses involving normal pollination. Lower seed set with mentor pollen was deduced to result from the use of less viable pollen, approximately half the pollen having been killed by alcohol. Pollinations at the diploid level resulted in more hybrids than at the tetraploid level, perhaps because genes for male sterility produced higher female sterility in the tetraploids. The self-compatible stock produced more seeds, mostly selfs, than the self-incompatible stock, but produced more hybrids only in one cross, T. pratense L. × T. diffusum Ehrh. The use of male-sterile female parents reduced selfing but produced fewer hybrids than male-fertile female parents. Techniques of this study were designed to affect prefertilization barriers, but the lack of effect may indicate that postfertilization barriers in Trifolium are of greater importance.