Aim: This study aimed to explore the effects of HLA-DRB alleles on Polygonum multiflorum-induced liver injury (PM-DILI). Methods: An exploratory study was conducted on 11 patients with PM-DILI using major histocompatibility complex targeted sequencing, and the frequencies of HLA alleles were compared with those of healthy controls in the Han-MHC database. In an independent validation study, the HLA-DRB1 and DRB5 genotypes of 18 cases with PM-DILI were detected. The impact of these two alleles on liver biochemistry and the type of liver injury was analyzed. Results: In the exploratory study, the frequencies of HLA-DRB1*15:01 and HLA-DRB5*01:01 in patients were higher than those in healthy Chinese population (OR: 3.91, 95%CI (1.42-10.01), P = 8.47×10-3 for HLA-DRB1*15:01; OR: 7.58, 95%CI (3.00-19.60), P = 1.46×10-5 for HLA-DRB5*01:01). In an independent validation study, there was a significant difference in the frequencies of these two HLA alleles between patients with PM-DILI and the healthy Chinese population (OR = 3.01, 95%CI (1.48-6.13), P = 4.07×10-3 for HLA-DRB1*15:01; OR = 5.06, 95%CI (2.62-9.77), P = 7.00×10-6 for HLA-DRB5*01:01). Combined analysis confirmed that these two HLA alleles were significantly associated with PM-DILI (OR = 3.34, 95%CI (1.93-5.78), P = 6.60×10-5 for HLA-DRB1*15:01; OR = 5.90, 95%CI (3.52-9.89), P = 2.87×10-10 for HLA-DRB5*01:01). Furthermore, the levels of liver enzymes and the frequency of the cholestatic/mixed type of liver injury were increased in patients carrying these two alleles. Conclusions: HLA-DRB1*15:01 and HLA-DRB5*01:01 were associated with PM-DILI, and may affect the severity and phenotype of Polygonum multiflorum-induced liver injury.