Performance of the Abbott m2000 instrument system and the Abbott RealTime™ HIV-1 assay was evaluated using a panel of 37 group M (subtypes A–D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87 % , 78 % , and 81 % of samples, respectively. RealTime HIV-1 detected an additional 31 samples at <40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87 % and 89 % of values were within 0.5 log₁₀ copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by 2 log₁₀ copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced “hands-on” time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.