Selection within elite cultivars for the improvement of agronomic traits is assumed to be ineffective due to the belief that cultivars are highly homogeneous. The objective of this study was to perform single-plant selection at ultra-low plant density to investigate the presence of genetic variation for higher crop yield. Single plants from three elite soybean cultivars were grown in a honeycomb design using a plant spacing of 90 cm. At least 350 plants from each cultivar were evaluated. Single-plant selection for high yield per plant was performed and a total of 20 plants were selected from within each cultivar. The selected plants from the honeycomb trial were extensively evaluated in row-plot experiments across three years and many locations. Our data provided evidence of significant intra-cultivar variation for seed yield. We found lines that, across years and locations, out yielded significantly the original cultivar where selection was initiated. These results suggest that single-plant selection within elite gene pools can be successful in improving the seed yield and agronomic performance of elite cultivars.