Background <p>In soybean, seed coat pigmentation is regulated by post-transcriptional gene silencing (PTGS) of <i>chalcone synthase</i> (<i>CHS</i>) genes at the <i>I</i> locus. Although several components of the PTGS pathway have been implicated in this process, genetic evidence linking specific RNA silencing factors to seed coat pigmentation remains limited.</p> Results <p>We identified three independent brown seed coat mutants from an EMS-mutagenized population of the soybean cultivar Pungsannamul, which normally produces yellow seeds. Genetic segregation analysis demonstrated that the brown seed coat phenotype in all three mutants is controlled by a single recessive locus. Complementation tests revealed that the three mutants are allelic. Genetic mapping by bulk segregant analysis (BSA) using a SNP genotyping array localized the causal locus to a 2.83-Mb interval on chromosome 11 containing the <i>ARGONAUTE5</i> (<i>AGO5</i>) gene. Sequence analysis identified distinct mutations in <i>AGO5</i> in each mutant line, including a splice-site mutation and two missense substitutions within the conserved P-element Induced Wimpy Testis (PIWI) domain. Co-segregation analysis using derived cleaved amplified polymorphic sequence (dCAPS) markers confirmed a complete association between <i>AGO5</i> mutant alleles and the brown seed coat phenotype. Expression analysis revealed significantly elevated <i>CHS7</i> transcript levels in the seed coats of all three <i>AGO5</i> mutants compared with the wild type.</p> Conclusions <p>Our results provide genetic evidence that AGO5 plays a critical role in PTGS-mediated regulation of <i>CHS</i> expression and seed coat pigmentation in soybean, consistent with phenotypes of previously reported PTGS component mutants such as <i>dicer-like2</i> (<i>dcl2a/dcl2b</i>) double mutants.</p>