Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.