Target region amplification polymorphism markers were used to assess the genetic diversity and relationship among 151 worldwide collected faba bean (Vicia faba L.) entries (137 accessions maintained at the USDA–ARS, Pullman, WA, 2 commercial varieties and 12 elite cultivars and advanced breeding lines obtained from Link of Georg-August University, Germany). Twelve primer combinations (six sets of polymerase chain reaction) amplified a total of 221 markers, of which 122 (55.2%) were polymorphic and could discriminate all the 151 entries. A high level of polymorphism was revealed among the accessions with an estimated average pairwise similarity of 63.2%, ranging from 36.9 to 90.2%. Cluster analysis divided the 151 accessions into five major groups with 2–101 entries each and revealed a substantial association between the molecular diversity and the geographic origin. All 101 accessions in Group V are originated from China and 13 of the 15 accessions in Group II were from Afghanistan. Thirty-two individual plants were sampled from two entries to assess the intra-accession variation. It was found that the advanced inbred line (Hiverna/5-EP1) had very little variation (5.0%), while the original collection (PI 577746) possessed a very high amount of variation (47.1%). This is consistent with the previous reports that faba bean landraces have a high level of outcrossing in production fields and thus contain larger amount variation within each landrace. One implication of this observation for germplasm management is that a relatively larger population is needed in regeneration to mitigate the possible loss of genetic variation due to genetic drift.