Genomic applications of DNA-binding molecules require an unbiased knowledge of their high. affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA binding sites in the reverse orientation for polyamides containing beta/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing S'-WCGCGW-3' from 5 '-WGCGCW-3'. Overall, this study displays the power of high throughput methods to aid the optimal targeting of sequence specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.