Phylogenetic relationships in the USDA Vigna germplasm collection are somewhat unclear and their genetic diversity has not been measured empirically. To reveal interspecific phylogenetic relationships and assess their genetic diversity, 48 accessions representing 12 Vigna species were selected, and 30 gene-derived markers from legumes were employed. Some high-quality amplicons were sequenced. Indels (insertion/deletions) were discovered from the sequence alignments that were specific identifiers for some Vigna species. With regard to revealing polymorphisms, intron-spanning markers were more effective than exon-derived markers. These gene-derived markers were more successful in revealing interspecific polymorphisms than intraspecific polymorphisms at both the DNA fragment and sequence levels. Two different dendrograms were generated from DNA fragment data and sequence data, respectively. The results from these two dendrograms supported each other and showed similar phylogenetic relationships among the Vigna species investigated. The accessions clustered into four main groups and 13 subgroups. Each subgroup represents a subgenus or a species. Phylogenetic analysis revealed that an accession might be misclassified in our collection. The putative misclassified accession was further supported by seed morphology. Limited intraspecific genetic diversity was revealed by these gene-derived markers and/or sequences. The USDA Vigna germplasm collection currently consists of multiple species with many accessions further classified into specific subspecies, but very few subspecies of the total subspecies available exist within the collection. Based on our results, more attention should be paid to the subspecies, wild forms and/or botanical varieties for future curation in order to expand the genetic diversity of Vigna germplasm in the USDA collection.