The Lotus Retrotransposon 1 (LORE1) is used for genome-wide mutagenesis of the model legume Lotus japonicus. Characterization of the LORE1 insertion sites in individual mutant lines is critical for development and use of the resource. Here we present guidelines for use of the LORE1 reverse genetics resource and provide detailed protocols for insertion site identification and validation. For high-throughput identification of insertions in up to 9,216 pooled lines, the FSTpoolit protocol takes advantage of Splinkerette adapters, molecular barcoding, 2D pooling, Illumina sequencing, and automated data analysis using the freely available FSTpoolit software. Complementing the high-throughput approach, we describe a simplified sequence-specific amplification polymorphism (SSAP) protocol well suited for quick identification of insertion sites in a limited number of lines. Both the FSTpoolit and simplified SSAP protocols are generally applicable to insertion site identification in any insertional mutagenesis setup.