Glycoprotein B (gB) is involved in cell to cell transmission of human cytomegalovirus (HCMV) and may be a critical factor in tissue tropism and viral pathogenesis. We analyzed the distribution of the four known gB genotypes of HCMV in 99 HIV-positive patients, 29 patients had HCMV retinitis, and 70 patients had asymptomatic HCMV infection. DNA was isolated from blood, urine, and aqueous humor, and gB genotypes were determined by PCR and restriction analysis. Infections with gB type 1 were less frequent in patients with retinitis than in patients with asymptomatic HCMV infection (17% versus 37%; p = 0.05). Furthermore, the gB type was correlated with dissemination of infection. In patients with HCMV detected in only one compartment (blood or urine) the gB type 1 was found more frequently than in patients with HCMV detected in at least two compartments (p = 0.01). The data show that gB genotypes differ in their association with clinical disease, and indicate that the gB genotype may contribute to the course of HCMV infection.