Ten mutants have been reported at the k2 (tan saddle seed coat) Mdh1-n (mitochondrial malate dehydrogenase 1 null) y20 (yellow foliage) chromosomal region in soybean [Glycine max (L.) Merr.]. The precise genetic mechanism(s) responsible for generating these mutants is (are) not known. The objective of this study was to determine whether chromosomal instability exists at this region. We introduced the w4-m and Y18-m mutable systems into the three independent sources of tan saddle seed coat mutants, T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2). A total of 12 bright yellow mutants were isolated with tan saddle seed coat, malate dehydrogenase 1 null phenotypes. Of these, 11 were found in 11 F2 mutant families out of a total of 977 derived by crossing T239 (k2), T261 (k2 Mdh1-n), and L67-3483 (k2) with six lines suspected to contain active transposable elements. One was found in the F3 generation derived from the cross A1937 x T239 (k2). Of the 11 F2 mutant families, 10 (out of a total of 381 F2 families) were associated with the T239 (k2) genetic background, and one out of 323 was associated with the T261 (k2 Mdh1-n) genetic background. But no mutation events were found among the 273 families with the L67-3483 (k2) genetic background. Allelism and inheritance studies indicated that these 12 bright yellow mutants were new mutants in the k2 Mdh1-n y20 chromosomal region. Thus, on introducing the w4-m and Y18-m mutable systems into T239 (k2) and T261 (k2 Mdh1-n) genetic backgrounds, chromosomal instability was induced in this region. In addition, 21 greenish yellow mutants were identified in the total of 977 F2 families. All 21 greenish yellow mutants were associated with the T239 (k2) genetic background. The mutations for greenish yellow foliage affected foliage color only at the seedling stage. Cosegregation of the tan saddle seed coat character with greenish yellow foliage were observed for these 21 greenish yellow mutants, suggesting that the greenish yellow phenotype may be due to a pleiotropic effect of the k2 allele in T239 or to chromosomal rearrangements at or near the k2 allele in T239. Finally, we believe that the genetic mechanism responsible for this high frequency of instability at the k2 Mdh1-n y20 chromosomal region involves receptor element activities present at this chromosomal region, which may contain complex chromosomal rearrangements in T239 and T261.