Genetic markers and chromosome linkage maps are useful in genetic research and plant breeding. Soybean [Glycine max (L.) Merr.], plants were grown in the greenhouse and field to obtain F1, F2, and F3 seeds, and starch gel electrophoresis was employed to determine inheritance of several aconitate hydratase isozyme variants and the relative position of the Aco3 locus in soybean Linkage Group 1. Genetic data show that the Aco1 null variant (aco1-n) is allelic to Acol-a, the Aco2-c variant is allelic to Aco2-a, and the Aco4-d variant is allelic to Aco4-c. The Aco5 locus has three alleles Aco5-a, Aco5-b, and aco5-n. Recombination values were 12.4 +/- 0.8% between the Aco3 and Sp1 (Am3), 33.6 +/- 3.7% between Aco3 and y12, 39.2 +/- 2.1% between Aco3 and t, 13.2 +/- 1.2% between Sp1 and y12, and 29.6 +/- 1.3% between Sp1 and t. The gene order of these markers was thus determined as Aco3-Sp1-y12-t. Based on these five aconitate hydratase isozymes, genetic variation was calculated for both wild and cultivated soybean. No significant differences in the level of genetic variation [gene diversity (heterozygosity) and number of alleles per locus] between wild and cultivated soybean were observed. The values for gene diversity and number of alleles per locus in soybean are comparable to those observed in other autogamous annual plant species.