Legumes constitute one of the most important global groups of agricultural species, providing a major source of protein and oil for humans and animals as well as fixing nitrogen and improving the fertility of soils. Gene technology can assist plant improvement efforts in clovers (Trifolium spp.), aiming to improve forage quality, yield, and adaptation to biotic and abiotic stresses. An efficient and reproducible protocol for Agrobacterium-mediated transformation of a range of Trifolium species, using cotyledonary explants and different selectable marker genes, is described. The protocol is robust and allows for genotype-independent transformation of clovers. Stable meiotic transmission of transgenes has been demonstrated for selected transgenic clovers carrying single T-DNA inserts recovered from Agrobacterium-mediated transformation. This methodology can also be successfully used for ‘isogenic transformation’ in clovers: the generation of otherwise identical plants with and without the transgene from the two cotyledons of a single seed.