BackgroundAfter the ABO (ABO) and Rh (RHD and RHCE) blood group systems, Kell (KEL), Kidd (SLC14A1), and Duffy (DARC) represent the second most important clinically relevant antigens.Study Design and MethodsSamples from 4000 Swiss blood donors, with serologic prevalues for K/k, Kpa/b, Jka/b, and Fya/b, and 48 additional samples of presumptive black African origin were genotyped using high‐throughput matrix‐assisted laser desorption/ionization, time‐of‐flight mass spectrometry, applying one single‐multiplex polymerase chain reaction/primer‐extension reaction simultaneously detecting 15 single‐nucleotide polymorphisms.ResultsGenotype/phenotype concordance for K/k, Kpa/b, Jka/b, and all Fya/b specificities were 100, 99.98, 99.93, and 99.20%, respectively. Discrepancies were caused by erroneous serologic profiles (n = 33), mainly attributed to weakly expressed Fyx (n = 28). Only three discrepancies had a genetic basis. They could all be explained by newly observed silenced alleles: one KEL*02N.34 and one FY*02N.03 with predicted R700Q and G261R amino acid exchanges, respectively, and one JK*B, with an as‐yet‐unidentified silencing cause. According to NCBI SNP database entry for rs8176034, another new allele, KEL*02.38, had been expected, and we formally demonstrated its presence. We furthermore identified individuals with rare phenotypes, such as Jsa/b heterozygotes among Caucasians, rare alleles, the “Swiss” JK*01N.03, and rare genotypes, such as one Fyx homozygote.ConclusionGenotyping proved its practicability in the daily routine setting and qualitatively outperformed serology. Technology is ideal for time‐insensitive donor genotyping and allows for a broad range of throughput needs. Consequently, from a technologic point of view, serotyping should be replaced by genotyping for donors' blood groups encoded by KEL, SLC14A1, and DARC.