Yam beans (Pachyrhizus DC.) are legume root crops that have attracted scientific interest because of high contents of starch, protein, and iron in their roots. In this study, 58 accessions of three cultivated Pachyrhizus species were characterized by amplified fragment length polymorphism (AFLP) molecular markers in order to estimate genetic diversity and interspecific relationships. To complement molecular marker information, individuals from each accession were analyzed in order to confirm ploidy levels. Eight AFLP primer combinations detected 136 (68.7 %) polymorphic bands. Shannon’s diversity indices (Hs) for each species were 1.04 (P. ahipa), 1.07 (P. tuberosus), and 2.42 (P. erosus), while the total diversity index was 2.45. Phylogenetic analysis, principal coordinate analysis and analysis of molecular variance (FST = 0.796) all showed significant species differentiation. All accessions were diploid (2n = 2x = 22), which is characteristic of the tribe Phaseoleae. Finally, a misclassified accession of P. tuberosus was identified. Molecular characterization of accessions is necessary for efficient management of germplasm collections.