Accumulating lines of evidence indicate that reactive oxygen species (ROS) are important signalling molecules for various cellular processes. 8-Oxo-7,8-dihydroguanine (OG) is a prominent oxidative modification formed in DNA by ROS. Recently, it has been proposed that OG may have regulatory and possibly epigenetic-like properties in modulating gene expression by interfering with transcription components or affecting the formation of G-quadruplex structures. Deciphering the molecular mechanisms of OG on regulation of gene expression requires uncovering the location of OG on genome. In the current study, we characterized two commercially available DNA polymerases, Bsu DNA polymerase (Bsu Pol) and Tth DNA polymerase (Tth Pol), which can selectively incorporate adenine (A) and cytosine (C) opposite OG, respectively. By virtue of the differential coding properties of Bsu Pol and Tth Pol that can faithfully or error-prone copy a DNA strand carrying OG, we achieved quantitative and single-base resolution analysis of OG in synthesized DNA that carries OG as well as in the G-rich telomeric DNA from HeLa cells. In addition, the parallel analysis of the primer extension products with Bsu Pol and Tth Pol followed by sequencing provided distinct detection of OG in synthesized DNA. Future application of this approach will greatly increase our knowledge of the chemical biology of OG with respect to its epigenetic-like regulatory roles.