Lotus corniculatus L. is the most important agricultural species in the genus Lotus and is the most widely distributed Lotus species worldwide. L. corniculatus genotypes form complex groups that are difficult to recognize both morphologically and biochemically. Given the extensive and expensive process of isolating Simple Sequence Repeats (SSR, also called microsatellites), the possibility of using microsatellites already identified in related species is highly attractive. The aim of this work was the identification and validation of transferable microsatellite markers in L. corniculatus, and using those markers to study the genetic variability among four cultivars. Each cultivar of L. corniculatus was represented by 15 genotypes. Ten microsatellite markers were evaluated, and from those, four were selected based on their discriminative values observed among cultivars. We detected 29 alleles for the four markers, and there was an average of 7.25 alleles per locus. The marker TM0197 had the fewest number of alleles (5) and TM0083 had the highest number of alleles (10). The polymorphic information content (PIC) for the selected markers varied from 0.19 to 0.35, and the markers were therefore classified as highly informative. Based on the markers, we found high variability between individuals of the same cultivar. The use of transferable microsatellite markers could be useful to differentiate individuals at a relatively low cost, showing a great potential for use in breeding programs.