Subterranean clover (Trifolium subterraneum L.) is widely grown for its forage and ability to fix atmospheric nitrogen. Development of new varieties is constrained by the slow turnover time of generations, with only one generation per year possible under field conditions. We present an in vitro-assisted single-seed descent (IVASSD) technique, which enabled turnover of 2.7–6.1 generations per year across a diverse range of 27 T. subterraneum cultivars encompassing subspeciessubterraneum, yanninicum and brachycalycinum. The IVASSD protocol accelerated the generation cycle in two ways: (i) time to floral initiation was minimised by growth under controlled temperature and extended photoperiod; and (ii) the seed-filling period was truncated and embryo and seed-coat dormancy avoided by the in vitro germination of immature seed on B5 medium plus L6KK overlay (0.525 mg gibberellic acid and 1.5 mg indole-butyric acid L–1). For the first time, an IVASSD system was validated on a full-scale breeding population with the production of 175 F7 recombinant inbred lines from an F4 population in less than one year. All F7 plants obtained were morphologically normal and fertile.