BACKGROUND Viral resistance to antiretroviral therapy threatens our best methods to control and prevent HIV infection. Current drug resistance genotyping methods are costly, optimized for subtype B virus, and primarily detect resistance mutations to protease and reverse transcriptase inhibitors. With the increasing use of integrase inhibitors in first-line therapies, monitoring for integrase inhibitor drug resistance mutations is a priority. We designed a universal primer pair to PCR amplify all major group M HIV-1 viruses for genotyping using Illumina MiSeq to simultaneously detect drug resistance mutations associated with protease, nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, and integrase inhibitors. RESULTS A universal primer pair targeting the HIV pol gene was used to successfully PCR amplify HIV isolates representing subtypes A, B, C, D, CRF01_AE and CRF02_AG. The universal primers were then tested on 62 samples from a US cohort of injection drug users failing treatment after release from prison. 94% of the samples were successfully genotyped for known drug resistance mutations in the protease, reverse transcriptase and integrase gene products. Control experiments demonstrate that mutations present at ≥ 2% frequency are reliably detected and above the threshold of error for this method. New drug resistance mutations not found in the baseline sample were identified in 54% of the patient samples after treatment failure. 86% of patients with major drug resistance mutations had 1 or more mutations associated with drug resistance to the treatment regimen at the time point of treatment failure. 59% of the emerging mutations were found at frequencies between 2% and 20% of the total sequences generated, below the estimated limit of detection of current FDA-approved genotyping techniques. Primary plasma samples with viral loads as low as 799 copies/ml were successfully genotyped using this method. CONCLUSIONS Here we present an Illumina MiSeq-based HIV drug resistance genotyping assay. Our data suggests that this universal assay works across all major group M HIV-1 subtypes and identifies all drug resistance mutations in the pol gene known to confer resistance to protease, reverse transcriptase and integrase inhibitors. This high-throughput and sensitive assay could significantly improve access to drug resistance genotyping worldwide.