Four sunflower species, Helianthus annuus, H. argophyllus, H. debilis and H. tuberosus, were characterized at the molecular level using the plastid trnH-psbA intergenic spacer. The trnH-psbA sequence was selected with the aim of developing a “DNA barcode” system (Kress et al., 2005) as a tool for species and specimen identification. The plastid region was PCR amplified with specific primers and sequenced with an ABI Prism 3730 Automated DNA sequencer. Intraspecific and interspecific sequence variation was evaluated to assess the resolution of the technique. Sequencing of both forward and reverse strands allowed for a high base calling accuracy and overcame the problem of polymerase slippage within microsatellite regions. After sequence editing, a very low (or absent) intraspecific variability was detected, whereas interspecific variability due to SNPs, indels and SSR length was sufficient for an unambiguous identification of each species.