The determination of HIV drug resistance mutations (DRMs) towards antiretroviral (ARV) drugs among HIV-1 treated patients with virological failure is crucial for further management of the patient. This study aimed to assess the most common genomic mutation and to analyse subtypes among the HIV-1 patients with viral load level > 1,000 copies/mL. A total of 101 virological failure HIV-1 patients from four different regions of Peninsular Malaysia with a viral load measurement facility were included in the study. Majority of patients (89.1%) have at least 1 mutation associated with clinical resistance to either protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs). Major resistance mutations among the patients towards NRTIs and NNRTIs were 70.3% and 18.8%, respectively. The most common mutation for NRTIs was M184V while K103N mutation was detected in the majority of patients who were treated with NNRTIs. The most commonly observed mutations for major PI and minor PI seen among the study population were V82A/T and L10V, respectively. In HIV-1 subtype analysis, CRF33_01B was the most predominant HIV-1 subtype in this study group. The vast detection of DRMs in this study emphasized the importance of genotypic resistance test in the management of HIV patients as DRMs can alter patient’s susceptibility towards ARV drugs. Further study on larger number of samples is essential for the development of a database on HIV-1 DRMs among patients that experience virological failure in Malaysia.