Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma. Display omitted •AID activity on single-strand DNA per se shapes the CDR-preferential SHM profile•Non-coding function of DNA at the mesoscale level directs AID deamination in vivo•Flexibility of single-strand DNA substrate contributes to AID substrate preference•DNA alteration can make a cold FR hot Somatic hypermutation in antibody-coding sequences preferentially occurs within specific regions; this study uncovers that the intrinsic single-stranded DNA flexibility within the regions confers the specificity.