Diverse variation of HIV-1 is a grave challenge for prevention of viral infection and immunotherapy. Monoclonal antibody (mAb) 2F5 recognizing an epitope ELDKWA (aa669-674) on HIV-1 envelope protein gp41 showed broad neutralizing activity against a lot of HIV-1 strains including primary isolates. However, viral mutation from ELDKWA to ELDEWA resulted in viral evasion from neutralization by mAb 2F5. Using ELDEWA-epitope-peptide (C-GFLDEWAGELDEWA) conjugated with carrier protein keyhole limpet hemocyanin (KLH), a mAb 14D9 (IgGl) was prepared and identified as the mAb with predefined ELDEWA-epitope specificity. The mAb 14D9 recognized the ELDEWA epitope, but not other three epitopes (ELDKWA, ELNKWA and ELEKWA). In comparison, mAb 2F5 could recognize only ELDKWA, but not three neutralization-resistant epitopes (ELDEWA, ELNKWA and ELEKWA). Interestingly, we searched several authoritative HIV sequence databases (http://hiv-web.lanl.gov) and found out that nearly all the viral isolates bearing the ELDEWA epitope belong to the O clade, the only exceptional viral isolate bearing the epitope ELDEWA has been demonstrated to be an intergroup M/O recombinant, which suggests that the ELDEWA-epitope on gp41 represents a specific epitope-marker of HIV-1 O clade. To confirm whether the mAb 14D9 recognizes gp41 of HIV-1 O clade, the rsgp41(IIIB), bearing ELDKWA-epitope was site-directed-mutated to the rsgp41 bearing ELDEWA-epitope. The mAb 14D9 could bind to rsgp41 bearing ELDEWA-epitope in immunoblotting analysis, did not bind to rsgp41 bearing ELDKWA-epitope. These experimental results suggest that the mAb 14D9 with predefined ELDEWA-epitope specificity may be applied to HIV-1 O clade identification.