LTR retrotransposons are closely related to retroviruses and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640,653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) ( http : // lotus .au . dk). Insertion preferences are critical for effective gene targeting, and we exploit our large data set to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134,682 Lotus japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research. This article is protected by copyright. All rights reserved.