Studies were conducted to (i) identify molecular probes that have a high probability of detecting polymorphisms among soybean [Glycine max (L.) Merr.] genotypes, (ii) determine the potential for analyzing genome structure using a molecular genetic map, and (iii) demonstrate the feasibility of a molecular pedigree analysis using mapped RFLP markers. Genomic DNA of 51 genotypes representing U.S. midwestern soybean germplasm was digested with the restriction enzymes HindIII, Dral, EcoRI, EcoRV, and TaqI. A random sample of 32 probes detected polymorphisms between paired comparisons of the 51 genotypes with a frequency from 0 to 72%. On average, no enzyme was strikingly better than any other for detecting polymorphisms. Homeologous chromosomal segments were detected, although they were clearly only remnants with recombination distance disparities and many rearrangements. The soybean genome, if indeed an ancient tetraploid, appears to be highly shuffled or diploidized. RFLP data were compared with the pedigrees of the cultivars Harosoy, Corsoy, Williams, and Clark, using from 58 to 105 mapped markers. Markers on Linkage Groups A and E were analyzed in detail to determine the map positions of polymorphisms and to identify chromosomal segments involved in cultivar development. The polymorphism percentage as detected by molecular markers coincided with genetic distance expectations based on pedigrees. These studies have demonstrated that probe-enzyme combinations can be identified that will be applicable to discerning differences among a broad range of genotypes. Additionally, it has been demonstrated that a detailed RFLP map can be used to increase our understanding of genome evolution and genome structure and, when combined with pedigrees, can be used to determine the ancestral sources of markers in a given cultivar's genome.