In soybean, dominant alleles of the I locus inhibit pigmentation of the seed coat while the homozygous recessive i allele results in fully pigmented seed. Previous work showed that dominant alleles correspond to duplications of chalcone synthase (CHS) genes leading to production of CHS siRNAs which in turn degrade CHS mRNAs resulting in yellow seed coats (Tuteja et al. Plant Cell 21, 2009). However, it was not known if the same phenomenon applied to the yellow and pigmented regions within the same seed coats that have homozygous i-i or i-k alleles that restrict pigment to the hilum and saddle regions of the seed coat, respectively. Here we describe the results of Illumina high throughput sequencing of small RNA populations from pigmented and yellow regions within seed coats with the same genotype. The level of CHS siRNAs is much higher in the yellow versus the pigmented region. CHS siRNAs from another genotype (i-i, k1), which also produces a pigmented saddle on the seed coat, are also much more abundant in the yellow versus the pigmented region. Small RNA blots show CHS siRNAs accumulated only in the yellow region and confirm the sequencing data. Thus, these data demonstrate that CHS siRNAs result in pigment pattern formations on soybean seed coats. In addition, small RNA sequencing data from twenty-one samples in six different tissues including cotyledons, roots, stems, and leaves showed that CHS siRNA expression is limited to the yellow seed coats confirming the tissue specificity of the generation of the CHS siRNAs.