Recombinant adeno-associated virus (rAAV) gene therapies rely on inverted terminal repeats (ITRs) for effective vector production and long-term efficacy. However, ITRs in plasmids used for rAAV production degrade during standard propagation in E. coli. Typically, 130 bp ITRs or even shorter variants instead of the full 143 to 145 bp ITRs are found. The implication of these truncated ITRs for vector production and quality has so far remained elusive. To solve this mystery, we first developed an amplification-free sequencing technique to assess ITR degradation on individual E. coli plasmid molecules. We then recovered full-length ITRs from rAAV and investigated their degradation using different E. coli strains and cultivation parameters. We found that cultivating E. coli Stbl3, DH5alpha, BL21(DE3), or JM103 at 42°C, instead of traditional temperatures (37°C or 30°C), stabilized these full-length ITRs in all tested cases. Cultivation at 42°C additionally enabled us to assemble and clone full-length ITRs from synthetic oligonucleotides. We suspected the ‘structural maintenance of chromosome’ proteins SbcC or SbcD as possible culprits for ITR degradation. Indeed, a ΔSbcC strain, JW0387-KC, was best at maintaining full-length ITRs at 37°C, while still profiting from cultivation at  42°C, where degradation was not detected. Importantly, using plasmids with these intact ITRs for rAAV production significantly increased genomic titers by up to 7-fold compared to standard ITR plasmids, while reducing reverse-packaged plasmid backbone. Our findings underline the importance of maintaining full-length ITRs and open the possibility to further enhance rAAV gene therapies through increasing ITR “health”.