Herbarium voucher specimens are indispensable for taxonomic research and one of the most important basic materials in biological research generally. However, depending on the condition of the specimen, it may be difficult to identify or effectively exploit the material for study. The DNA barcoding technique is an important research tool for identification based on specimens or samples using standard DNA sequences, and has become much more widely used in recent years. Here, we make a case study for identification of incomplete or otherwise difficult specimens using two imperfect plant specimens deposited in the herbarium HIRO by means of the DNA barcoding technique. DNA barcoding showed that one of the subject specimens from Okinawa Prefecture, southernmost Japan, could be identical to Ruellia longepetiolata (Oerst.) Hemsl. (Acanthaceae). Using a BLAST search, sequences obtained from the other specimen identified only as Symplocos sp. (Symplocaceae), also from Okinawa Prefecture, did not match any sequences deposited in the DNA database, suggesting that further studies would be needed based on traditional taxonomic methodology. The results demonstrated that (1) DNA barcoding is a useful tool to identify specimens in poor condition in the herbarium, (2) phylogenetic analyses are helpful in to determining the identity of specimens, and (3) identification using only DNA barcoding has limitations and is dependent on the extent of the DNA library.