Alfalfa, the world’s most important forage legume, is also Wyoming’s most important crop. Most alfalfa cultivars are bred as synthetics. Typically, selected clones are intermated (either by insects or by hand) to produce Breeder Seed, the SYN1 generation. Subsequently, SYN2 (Foundation Seed) and SYN3 (Certified Seed; commercial seed) generations are produced in the field via insect pollination. Because alfalfa is an autotetraploid (2n=4x=32), it is possible that higher levels of heterozygosity in the SYN1 generation may transmit that heterozygosity (via diploid gametes, n=2x=16) to progeny resulting in higher levels of heterosis (hybrid vigor) in subsequent generations thereby reducing inbreeding depression due to intermating of related plants. Our study tested the hypothesis that more frequent shifting from plant to plant during “chain-crossing” of parental clones may result in higher levels of outcrossing. Specifically, we crossed the same 42 plants by (1) tripping 6 florets on each plant before moving to the next plant, and (2) tripping 15 florets per plant before moving on. Results indicate that the first method produced significantly more seeds per pod, with self-pollinated checks producing very low numbers. We speculate that this is due to a higher level of cross-pollination and a lower level of self-pollination when the shift from one parent to the next is more frequent. In addition, because the 42 plants came from two different populations, we also performed intra-population chain crosses that produced less seed per pod than the interpopulation crosses, demonstrating interpopulation heterosis. Different potential methods for “frontloading heterozygosity” in alfalafa will be also be discussed.