Human immunodeficiency virus type 1 (HIV-1) genotypic susceptibility testing is an important procedure in the treatment of HIV-1 infection because it provides the information required for optimizing antiretroviral drug regimens. The reliability of this test depends on the accuracy of the software used to interpret the genotypic data. Although there are nine different genetic HIV-1 subtypes and over 20 circulating recombinant forms (CRFs) accounting for the majority of cases in the AIDS pandemic reported to date, most current interpretation software detects viral gene mutations by merely comparing sample sequences with the HIV-1 subtype B reference strain sequence regardless of the sample subtype. We hypothesized that proper selection of a reference sequence corresponding to the patient’s viral subtype for detecting gene mutations would provide a more appropriate input for genotypic interpretation.