There was an outbreak of HIV-1 transmission among 20 out of 122 Korean hemophiliacs from 1990. We assessed the genetic relationships among HIV-1 viruses found in three cash-paid plasma donors whose preseroconversion plasma was used to produce Korean-made clotting factor, 20 hemophiliacs infected with HIV-1 in Korea, three hemophiliacs infected with HIV-1 from clotting factor manufactured outside Korea, and 71 local control patients infected with the Korean subclade of HIV-1 subtype B (KSB). Full-length pol gene sequences (2841 bp) of viruses from frozen stored serum, samples obtained 1-3 years after diagnosis, were amplified by RT-PCR and sequenced by direct DNA sequencing. Phylogenetic and signature pattern analyses were used to investigate the relationships among the sequences. Donors O and P were associated with two clusters, of 8 and 12 hemophiliacs, respectively, which were demarcated from the 71 KSB-infected local control patients and donor R. These data confirm that HIV-1 transmission to 20 hemophiliacs occurred through infusion of Korean-made clotting factor.