We report the proof of concept of a novel DNA sequencing technology called sequencing by denaturation (SBD). SBD is based on the Sanger sequencing reaction performed on amplified target templates immobilized on a solid surface followed by the denaturation of these Sanger fragments. As these fluorescently labeled fragments denature sequentially, the fluorescence intensities in the four channels corresponding to the four base types are monitored in a flow cell. A sequencing instrument with a microfluidic flowcell has been custom-designed to integrate automated fluidics, temperature control, and fluorescence imaging. The denaturation profiles of several synthetic oligonucleotides were measured with this system and our data demonstrated the ability to sequence short DNA by SBD. SBD is a simple and fast method with the potential to improve the speed and cost of large-scale genome re-sequencing.