Breeding programs in major crops normally restrict the use of parents to those improved for a variety of traits. As favorable alleles are selected and fixed, there is concern among the breeders that genetic variation may be reduced. On the other hand, continuous genetic gain within elite gene pools suggests that the genome is more plastic to selection than previously assumed. This study investigated the presence of genetic variation for seed weight, seed protein and seed oil in advanced generations of selfing within three elite soybean cultivars. Single-plant selection within each soybean cultivar was performed for two years using a honeycomb design and a plant spacing of 1.4 plants/m2. The selected single-plant progeny lines were further evaluated in replicated row-plot experiments across 3 years. The results showed that divergent honeycomb selection for seed protein, oil, and seed weight was successful in identifying significant variation within the F17 generation. This study is the first one to report the existence of genetic variation in advanced generations of selfing in soybean and it suggests that such variation may be constantly created in response to various environmental stimuli.