summary.In screening 87665 unrelated healthy blood donors in China, serology studies resulted in the detection of two K0 probands, both female. To explore the molecular basis of the Knull phenotype in the Chinese population, genomic DNA, total RNA, and reticulocyte RNA were subsequently prepared from the two probands, five family members of proband 1, four unrelated normal controls, and one unrelated KEL1 control. Nucleic acids were analyzed for the KEL gene by DNA and RNA sequencing, while antigens were analyzed by flow cytometry with BRIC18, BRIC68, anti‐k, and anti‐Kpb. Two novel Knull alleles were identified in both probands: in exon 3, 185insT (Ser62Phe and a premature stop codon in exon 4, GenBank accession number, EF208900), and in exon 7, 715G>T (Glu239Stop, GenBank accession number EF208901). Alternative splicing patterns were observed in RNA obtained from whole blood versus from a reticulocyte fraction. Our study identified these two novel Knull alleles resulting in the Knull phenotype, the frequency of the Knull phenotype amongst Chinese mainlanders is only 0.00228%.