An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV-beta-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM-mediated restriction mapping of DNA these mutations can be positioned relative to EcoRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA.