Environmental stimuli can generate intra-cultivar genetic variation in nearly homozygous soybean cultivars. This genetic variation may be useful for enhancing genetic diversity and developing new cultivars. Soybean plants exposed to multiple environmental stimuli produce seed variants with different phenotypic and genotypic characteristics. When these variants are selfed, the variation is heritable and stable. Seed produced in plants subjected to wide-spaced honeycomb (HC) planting, natural hail and to a modified accelerated aging (AA) at planting has exhibited intra-cultivar genetic variation. However, the effect of a single environmental stimulus on agronomic performance, seed characteristics and stability of variants from a soybean plant and its progeny had not been evaluated. The main objective of the present study was to evaluate agronomic performance, seed characteristics and aconitase-4 (Aco4) isozymes allele variability and stability in progenies of soybean cultivars and variants exposed to environmental stimuli. The stimuli included the wide-spaced HC planting design, simulated hail and modified AA. Progenies from soybean plants grown in wide spaced HC planting design had greater variation in agronomic performance and seed characteristics than those from conventional-row planting and modified AA. Simulated hail treatment did not have a significant effect on agronomic performance. Progenies from variants had greater variability and performed better for most agronomic performance and seed characteristics than those from soybean original cultivar. New double and single Aco4 isozyme allele switching were identified in one seed within a three-seeded pod of the variants grown in a wide space HC planting design. DNA sequence data of the Aco4 gene (Glyma.11g080600) of these new variants indicated single nucleotide polymorphisms (SNPs) substitutions in the exons, which resulted in amino acid substitution and production of slightly different proteins. These studies confirmed that the wide-spaced HC planting design is a valuable tool for generating intra-cultivar genetic and phenotypic variation in soybean cultivars. Plant breeders could use wide-spaced HC planting design to induce novel genetic and phenotypic variation for new cultivar development.