ABSTRACTA method is described for obtaining interspecific hybrids between commercial lines of Lotus uliginosus Schkuhr and Lotus corniculatus L. Hybridization was possible between these species using embryo rescue. Two strategies were used to confirm 40 F1 hybrids, that is, flow cytometry and microsatellite simple sequence repeats (SSRs). The L. uliginosus and L. corniculatus parents have significantly different genome size (2.5 ± 0.02 and 2.22 ± 0.02 pg, respectively) and the offspring from the cross had intermediate values. Two SSR primers were polymorphic between the genotypes tested, TM1150 and EH380069. Maternal effect was detected in L. uliginosus genotypes, as determined by greater frequency of F1 hybrids. Recombinant traits were observed in the F1 hybrid progeny. Ninety F1 hybrids resulting from L. uliginosus × L. corniculatus crosses showed different levels of fertility in the polycross; <3% F1 plants produced more than 1000 seeds without embryo rescue while 10% of plants did not flower the first year. The F1 hybrid plants grown under greenhouse conditions exhibited shoot phenotypes similar to L. uliginosus (maternal) whereas F2 phenotypes were similar to L. corniculatus (paternal) in field trials. Rhizome presence was observed in 60% of F2 hybrid plants. The diameters of the root crowns of these plants, however, were similar to that of L. corniculatus.