Objectives: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. Methods: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score=10 to at least one InSTI. To rule out circulation of minority InSTIresistant HIV, 65 samples were selected for 454 integrase sequencing. Results: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIswere detected. Eleven (4%) subjects hadmutations at resistance-associated positions with an HIVdb score =10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutationsweredetected, whereas integrase substitutionswithanHIVdbscore=10were found in8(14.3%) individuals. Conclusions:No signature InSTI-resistant variantswere circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistancewere not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.