Abstract: The present invention describes a method for separating heteroduplex and homoduplex DNA molecules in a mixture. In the method, such a mixture is applied to a stationary reverse phase support. The heteroduplex and homoduplex molecules are eluted with a mobile phase containing an ion-pairing reagent and an organic solvent. The eluting is carried out under conditions effective to at least partially denature the heteroduplexes (e.g., thermal or chemical denaturing) resulting in the separation of the heteroduplexes from the homoduplexes. The method has many applications including, but not limited to, comparative nucleic acid sequencing, linkage analysis, evolutionary studies, forensics, identification of disease-causing gene mutations, genetic marker development and diagnostics.