Two perennial Trifolium, T. polymorphum Poir. and T. argentinense Speg., are American clovers unique within the genus for being amphicarpic. There is no consensus in the literature regarding the breeding system of the aerial flowers of T. polymorphum, therefore, the breeding system was studied. In 1997 T. polymorphum was collected in Uruguay and evaluated at INIA Tacuarembó. In 2001, 10 field patches were marked and in 2004, 20 plants per patch were characterized with simple sequence repeat markers. Patch J10 showed a particular molecular profile, therefore, 198 open-pollinated progenies freely visited by honeybees were studied. In 2005, at the University of Florida, Gainesville, different hand-pollination treatments were conducted within an accession from Paraguay. Trifolium polymorphum was able to cross-pollinate with all the known pollen donors molecularly marked that surrounded plants from patch J10 (30%), also with some nonmarked native ones from the vicinity (10%) as well as with itself (60%), when allowed to be visited by honeybees. However, when there are no pollinators, the selfing rate is minimal. The proposed classification for the breeding system is an allogamous, self-compatible species that benefits from pollinators to set seed.