BACKGROUND:: The IPERGAY ANRS trial showed that on-demand pre-exposure prophylaxis (PrEP) with tenofovir (TDF) and emtricitabine (FTC) was highly effective in preventing HIV infection among highly exposed men who have sex with men. Here we analyzed drug resistance-associated mutations (RAMs) among all participants who acquired HIV infection during this trial. METHODS:: Resistance was analyzed on frozen plasma at the time of HIV diagnosis among participants enrolled in the double-blind and open-label phases of the ANRS IPERGAY trial. Adherence was measured by pill counting and by plasma tenofovir and FTC assay. Reverse transcriptase (RT) sequencing was performed, using population-based and ultradeep sequencing (454 GS Flex). RESULTS:: During the trial, 31 participants were diagnosed with HIV-1 infection (subtype B, 64.5%), using antigen/antibody immune assay in 29 cases and plasma HIV RNA assay in two. The median plasma HIV-1 RNA level was 5.52 log10 copies/ml. Twelve participants were tested for drug resistance before starting PrEP (6 assigned to TDF/FTC, 13 to placebo). Primary resistance to nucleoside RT inhibitors (zidovudine) and/or non-nucleoside RT inhibitors was detected in 6 participants (19%; 95% CI: 7 – 42). No major or minor TDF- or FTC-resistant variants were detected. CONCLUSION:: No TDF or FTC resistance-associated mutations were found among participants who acquired HIV in the ANRS IPERGAY trial.