We developed a polymerase chain reaction (PCR) method, based on strain-to-strain variation of DNA repeats in the herpes simplex virus type 2 (HSV-2) genes US1 and US12 to genotype HSV-2 strains and determine the incidence and risk factors associated with HSV-2 superinfection in patients with recurrent genital herpes (RGH). Forty-seven (92%) of 51 unrelated HSV-2 isolates could be distinguished. Genotyping of sequential HSV-2 isolates showed a different genotype in all of the 11 human immunodeficiency virus type 1 (HIV-1)–seropositive patients with RGH, compared with 1 of the 8 HIV-1–seronegative patients with RGH. The PCR approach developed distinguishes unrelated HSV-2 strains efficiently and indicated a high incidence of genotype variance between sequential HSV-2 isolates of HIV-1–seropositive patients with RGH