SignificanceA common way of becoming resistant to antibiotics is mutations in the bacterial genome. However, how these mutations arise remains poorly understood. Here, we determine that endogenous oxidative stress is the main driver of mutagenesis that leads to antibiotic resistance development. In addition, we find that a highly conserved DNA repair pathway, transcription-coupled repair, is responsible for this oxidative stress-driven evolution of antibiotic resistance. Our prior work showed that the Mfd protein, which contributes to TC-NER, accelerates the development of antibiotic resistance. The work presented here shows that, in fact, all of the proteins involved in TC-NER are contributing significantly to resistance development, expanding our knowledge of the fundamental mechanisms driving not only drug resistance, but also evolution in general.