To investigate the molecular mechanism and distribution characteristics of RhD negative phenotypes in Han population of blood donors in Wuhu city. A total of 210 RhD- samples from August 2021 to August 2022 were screened by serological test and collected from Wuhu Central Blood Station for the voluntary blood donor population. Exons 1 and 10 of the RHD gene were amplificated by PCR to determine whether the samples had the RHD gene. Exons 1-10 of the RHD gene were amplificated by PCR and zygosity analysis were performed in 82 samples containing D gene, and Sanger sequencing was performed on 55 samples containing all RHD exons to determine the genotype. Among 210 RhD- specimens, 128 cases (60.38%) had RHD gene deletion. 27 cases had partial exons of RHD, including 2 cases with RHD*DVI.3/RHD*01N.01, 24 cases with RHD*01N.04/RHD*01N.01, and 1 case with RHD-CE(2-10)/RHD*01N.01. 55 cases had retained all of 10 exons, including 4 cases with RHD*01/RHD*01N.01, 6 cases with RHD*15/RHD*01N.01, 1 case with RHD*01W.72/RHD*01N.01, 1 case with RHD*15/RHD*01EL.01, 39 cases with RHD*01EL.01/RHD*01N.01, and the remaining 4 cases were determined to have no RHD gene deletion by zygosity analysis and sequencing showed the presence of 1227G>A mutation loci. There is polymorphism in the molecular mechanism of RhD- D gene in Wuhu blood donor population, among which RHD*01EL.01 and RHD*15 are the main variants in this region. The results of this study provide a theoretical basis for RhD blood group identification and clinical blood transfusion in this region.