A study of the genetic variability of herpes simplex virus (HSV) type 1 from recurrent lesions and clinical reinfections was done using restriction endonuclease analysis and the RNase A mismatch cleavage method. Comparative genetic analyses of HSV-1 recurrent isolates from 1 patient and of HSV-1 isolates from different anatomic areas (vagina and lip) from another patient showed differences only in the glycoprotein B gene but not in the thymidine kinase gene even though the viruses had the same restriction endonuclease pattern. These results suggest the RNase A mismatch cleavage method is useful for epidemiologic studies of DNA viruses.