DNA barcoding employs sequence variation in short, standardized gene regions as a tool to discriminate species and has many applications in plant authentication. Success amplification through PCR plays a vital role in DNA barcode library construction and sequencing. This study aims to improve determine the optimal annealing temperature for DNA barcode amplification. In this study, eight DNA barcode regions including ITS, matK, rbcL, rpoC1, ycf1b, trnH-psbA, atpF-atpH and psbK-psbI were amplified by gradient PCR to assess and determine the proper annealing temperature. Our results indicated that the PCR yield and specificity for ITS, matK, rbcL, rpoC1 and ycf1b were optimized using gradient PCR. 58°C was required for optimal primer binding temperature in ITS regions while the other regions involved lower annealing temperature, ranging from 49.1°C to 54.2°C. These findings illustrated that an appropriate annealing temperature contributed significantly for PCR success, which is a key step for sequencing quality.