Manual cross-pollination to produce large quantities of hybrid soybean seed for yield trials is very difficult and time-consuming. This has been one the reasons why parental combinations that produce heterosis levels superior to the best pure-line cultivars, (i.e., identification of heterotic patterns) have not been identified in soybean. However, insect-mediated cross-pollination has been shown to produce large quantities of hybrid soybean seed and would facilitate the identification of heterotic patterns in soybean. The objective of this study was to evaluate F₁ hybrid soybean seed from single–crosses, three-way crosses, and backcrosses (BC₁F₁) produced by selected male-sterile lines as female parents and a selected group of male parents. Bees were the pollinator vector. Eight cross-combinations were produced in a complete randomized block design with five replications in summer 2002. In summer 2003, the F₁ seed and their parental lines were evaluated for yield, height, lodging, seed protein, seed oil and seed fatty acids content at three locations Ames, IA, Gilbert, IA, and Otterbein, IN. In summer 2003, the BC₁F₁ and three-way crosses were produced. The BC₁F₁ seed was obtained from the plants of the original eight single cross-combinations as female parents backcrossed to their recurrent male parents. The three-way seeds were produced using the same original eight single cross-combinations as female parents and crossed to a common male parent (a high yielding commercial line).The BC₁F₁ , the three-way crosses, and their parental lines were evaluated in summer 2004. Results indicated that for the traits evaluated, positive and negative mid-parent heterosis was observed in the single-crosses, in the BC₁F₁ and in three-way crosses.