Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to assess the variability in tomato germplasm (Lycopersicon esculentum Mill.), which included 46 accessions from the following groups: vintage cultivars, modern cultivars, South American regional cultivars, and wild L. esculentum van cerasiforme. Two L. cheesmanii accessions served as an outgroup. The 48 accessions were monomorphic at 135 of the 215 RAPDs loci and 31 of the 48 RFLP loci that were assayed. Alleles were identified that distinguished the five groups and many of the cultivars. The frequency of polymorphic loci was low in vintage cultivars, with less than 2.8% of the loci being variable within the group. In contrast, introgression of wild germplasm into modern cultivars has increased the polymorphic loci to 11.6%, whereas within the group of regional cultivars linkage drag and outcrossing may be responsible for the further increase to 20.3%. These levels of genetic variability were lower in comparison to the 24.5% polymorphic loci of cerasiforme, a group that may contain unutilized desirable traits. The small genetic distance from the vintage cultivars to several of the widely distributed regionals and cerasiformes indicated that proximity of vintage cultivars in Latin America may have resulted in intercrossing of these materials with the wilder germplasm. RAPDs appear promising for both germplasm fingerprinting and as a predictor of genetic diversity for plant breeding applications.