Abstract: Background　Precision treatments are crucial for HBV infections and detection of HBV drug-resistant sites is a prerequisite. However, none of the available kits for detection of HBV drug-resistant sites are suitable for large-scale clinical application. Objective　To establish and evaluate the platform for detection of drug-resistant sites of hepatitis B virus (HBV) based on Sequenom MassARRAY SNP that combined single-base extension PCR, multiplex PCR, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods　A total of 100 HBV-positive patients who were admitted to the Fifth Medical Center of PLA General Hospital from 2014 to 2018 were randomly selected. The cohort included 51 males and 49 females, with a median age of 51 years. Sequencing was successfully completed in 62 cases. Sequence comparison and cluster analysis were performed using MEGA11.0. The polymerase gene sequences of wild-type and mutant-type of HBV were synthesized. Amplification primers and single base extension primers for 19 SNP sites of HBV were designed and synthesized. The corresponding Sequenom MassARRAY SNP platform was established to evaluate the detection limit of each resistance SNP. Eighteen clinical serum samples were randomly selected from 62 samples with confirmed full-length polymerase sequences, and then detected by sanger sequencing and Sequenom MassARRAY SNP. Results　Among the 62 HBV clinical samples, the genotype B and C accounted for 25.8% and 74.2%, respectively. The synthesized wild-type and mutant-type polymerase gene sequences covered a total of 15 drug-resistant sites (including 19 SNPs). The established Sequenom MassARRAY SNP detection platform showed that the detection limits of 19 SNPs were 1%-5%. No SNPs of HBV resistance were detected by Sanger sequencing among the 18 clinical serum samples, while 5 samples were detected to contain SNPs of HBV resistance by Sequenom MassARRAY SNP, including 4 genotype B and 1 genotype C.Conclusion　The Sequenom MassARRAY SNP-based drug-resistance detection platform for hepatitis B virus is established. The preliminary evaluation demonstrates that this platform has the characteristics of rapid accuracy, high throughput, and high sensitivity, which can determine the drug-resistant profiling of HBV in one experiment.