Objective Identification and sequence characteristics analysis of Atractylodes chinensis (DC.) Koidz. seeds were executed based on DNA barcoding technology, to explore the feasibility of the DNA barcoding technology for the authentication of Atractylodes chinensis (DC.) Koidz. seeds. Methods A total of 12 seeds of Atractylodes chinensis (DC.) Koidz. were collected, and the morphological features of seeds were described by stereomicroscope. The ITS2 and psbA-trnH sequences were obtained after DNA extraction, polymerase chain reaction (PCR) and bi-directional sequencing. The quality of chromatographic traces was evaluated using Codoncode Aligner V9.0.1, and multi-sequence alignment and mutation site analysis were performed by MEGA-X. Results The results of DNA extraction, PCR amplification and sequencing were successfully obtained with a rate of 100%, and the sequencing output files were high quality. Moreover, 12 ITS2 sequences and 12 psbA-trnH sequences of Atractylodes chinensis (DC.) Koidz. seeds were obtained, respectively. Among which, two insertion/deletion sites were found in ITS2 sequences, and 12 psbA-trnH sequences could be divided into four haplotypes, including three variable sites. It indicated that the intraspecific variation of Atractylodes chinensis (DC.) Koidz. seeds from different habitats was relatively conservative. Conclusion The research of DNA barcoding and sequence characteristics analysis based on ITS2 and psbA-trnH sequences can provide support for species identification and genetic diversity of Atractylodes chinensis (DC.) Koidz. seeds.