Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.