OBJECTIVES To assess the prevalence of resistance to rilpivirine and mutations at position 138 in reverse transcriptase and to identify associated epidemiological and biological characteristics. METHODS This retrospective study included 238 patients with available HIV-1 nucleotide sequences analysed at the Laboratory of Virology at the University Hospital of Nancy between January 2011 and June 2013. Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) was evaluated according to the ANRS algorithm (version 23) and correlated with clinico-epidemiological and therapeutic data. The virus strains were analysed by evaluating the distance and distribution of the phylogenetic tree (MEGAv5). RESULTS Among previously treated patients (111/238, 46.6%), 68/111 (61.3%) had received NNRTIs; all were rilpivirine-naive. The prevalence of rilpivirine resistance in the whole cohort was 12.6% (30/238), and was 10.2% (13/127) and 15.3% (17/111) in naive and pre-treated patients, respectively. The E138A mutation was the most frequent mutation associated with resistance to rilpivirine (P < 0.0001). The prevalence of the E138A mutation tended to increase over time, from 3.6% (2/55) during the first half of 2011 to 9.3% (4/43) during the first half of 2013 (P = 0.0614). Seven viral strains from seven naive male patients positive for the E138A mutation appeared in the same cluster. CONCLUSIONS In our cohort of patients, we observed significantly increased resistance to rilpivirine, mostly because of the E138A mutation, probably due to an E138A strain circulating in newly diagnosed men who have sex with men. Taken together, our results emphasize the need to investigate the prevalence of rilpivirine resistance-associated mutations in the coming years both in France and abroad.