Plant retrotransposons are among the largest transposon families. Tnt1, Tos17, Tto1 and LORE1 have been widely used in establishing mutant populations. In the present study, we summarized and compared various approaches for establishing mutant populations, especially Tnt1 retrotransposons. We focused on advances in establishing and using the Medicago truncatula Tnt1 mutant population, including nitrogen fixation, metabolism and development. Our study will benefit the comprehensive understanding and use of the M. truncatula Tnt1 mutant population and provide valuable references for researchers studying legume functional genomes.