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Abstract Because of the detrimental effects of terrestrial invasive plant species (TIPS) on native species, ecosystems, public health, and the economy, many countries have been actively looking for strategies to prevent the introduction and minimize the spread of TIPS. Fast and accurate detection of TIPS is essential to achieving these goals. Conventionally, invasive species monitoring has relied on morphological attributes. Recently, DNA‐based species identification (i.e., DNA barcoding) has become more attractive. To investigate whether DNA barcoding can aid in the detection and management of TIPS, we visited multiple nature areas in Southwest Michigan and collected a small piece of leaf tissue from 91 representative terrestrial plant species, most of which are invasive. We extracted DNA from the leaf samples, amplified four genomic loci (ITS,rbcL,matK, andtrnH‐psbA) with PCR, and then purified and sequenced the PCR products. After careful examination of the sequencing data, we were able to identify reliable DNA barcode regions for most species and had an average PCR‐and‐sequencing success rate of 87.9%. We found that the species discrimination rate of a DNA barcode region is inversely related to the ease of PCR amplification and sequencing. Compared withrbcLandmatK, ITS andtrnH‐psbAhave better species discrimination rates (80.6% and 63.2%, respectively). When ITS andtrnH‐psbAare simultaneously used, the species discrimination rate increases to 97.1%. The high species/genus/family discrimination rates of DNA barcoding indicate that DNA barcoding can be successfully employed in TIPS identification. Further increases in the number of DNA barcode regions show little or no additional increases in the species discrimination rate, suggesting that dual‐barcode approaches (e.g., ITS + trnH‐psbA) might be the efficient and cost‐effective method in DNA‐based TIPS identification. Close inspection of nucleotide sequences at the four DNA barcode regions among related species demonstrates that DNA barcoding is especially useful in identifying TIPS that are morphologically similar to other species.