We evaluated the sequence diversity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in HCV-infected patients in whom the mode of transmission is unknown. The sequence diversity of HVR1 in 26 Japanese patients with chronic HCV infection of unknown mode of transmission (UT) was compared with 17 patients with chronic posttransfusion hepatitis C in whom only a single HCV infection had occurred (PH), and with 18 patients with hemophilia with chronic HCV infection who might have been multiply infected with HCV (HE). The diversity of HVR1 was evaluated by direct sequencing after PCR amplification of HVR1. The sequence diversity of HVR1 was 10.1 +/- 7.7% in UT, 2.7 +/- 2.8% in PH, and 14.6 +/- 6.9% in HE. The diversity of the patients with unknown transmission was greater than that of the posttransfusion hepatitis patients, and in some patients it was similar to that of multitransfused hemophiliac patients (UT vs PH, P = 0.0004; UT vs HE, P = 0.04; and HE vs PH, P < 0.0001). Multiple infections with HCV could have occurred frequently in patients with chronic HCV infection in whom the mode of transmission was unknown, which increased the sequence diversity of HVR1 of these patients.