In Turkey, the first HIV/AIDS case was reported in 1985. Since then the number of persons with HIV infection has increased, HIV is getting a public health problem. The aim of this study was to determine HIV-1 subtype diversity, drug resistance and gag cleavage site mutations among 20 HIV-infected men having sex with men from Istanbul, Turkey. The most prevalent subtype was found to be subtype B (50 %), but also the non-B subtypes A1, C and CRF02_AG, CRF03_AB and CRF06_cpx were found. Resistance-associated mutations were found in 6 patients (30 %) with 2/6 patients being therapy-experienced and 4/6 therapy-na < ve at the time-point of analysis. In these patients, the nucleoside reverse transcriptase inhibitor (NRTI)-associated resistance mutations M41L, T215C, V75I, T69N, the non-NRTI associated mutations V106I, E138A, K103N and the protease inhibitor associated mutations Q58E and V82I were detected. Two virus strains also presented Gag cleavage site mutations. With increasing numbers of HIV-infected Turkish patients that require anti-retroviral treatment, HIV-1 drug-resistance testing is strongly recommended in order to choose the most active drug combination for therapy to achieve better clinical outcomes.