All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNAâbinding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific coârepressors (CoRs) they recruit have not been determined. Here, we develop a highâthroughput, nextâgeneration sequencingâbased screening method, repressiveâdomain (RD)âseq, to systematically identify RDs in complex DNAâfragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcriptionârelated proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, we show that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance our understanding of repressors, their sequences, and the functional impact of sequenceâaltering mutations and should provide a valuable resource for further studies.