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Background and Aims: Genotyping assay has been accepted as a guidance in the therapeutic management of Human Immunodeficiency virus 1 (HIV-1). But, it is not commonly used in our country due to its high running cost. The aim of this study is evaluate an in-house genotyping resistance test (GRT) for HIV-1. Methods: HIV-1 RNA of 20 samples were extracted from plasma and RT Nested- PCR was performed and the final products were sequenced. Stanford HIV Sequence Database was used for genotyping and interpretation of resistance data. Results: Subtype A was the dominant viral subtype in these patients. Also the results of drug resistance interpretation showed that drug-naïve patients are susceptible to drugs and for patients taking the drugs 66.6% susceptible for AZT, 50% high- level resistance for 3TC, 66.6% low- level resistance for ABC, 66.6% susceptible for TDF, 50% high- level resistance for NVP and 50% high- level resistance for EFV. Conclusion: Our method is able to amplify and sequence HIV-RNA from plasma samples from a random selection of patients encompassing different subtypes. The results of this study may have important consequences for survey and clinical management of patients with AIDS in Iran.