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Aim We have developed an allele-level resolution next generation sequencing (NGS) strategy for HLA class I alleles and used it to explore HLA diversity in a registry donor population. Methods Long-range amplification of full-length HLA-A, -B, -C loci was performed in separate polymerase chain reactions using DNA from a buccal swab. Amplicons from one individual were then pooled and sheared by sonication. A library was constructed using Illumina’s TruSeq Nano kit and the DNA fragments tagged with one unique index combination. Libraries from 96 individuals were combined and sequenced simultaneously in a single 500 cycle (V2) paired-end run using an Illumina MiSeq. Data analysis used Conexio Genomics AssignTM MPS software. Results The typing protocol was validated with 552 donor registry samples previously typed by Sanger sequencing and probe hybridization at single genotype G level resolution, yielding a concordance of 99%. NGS yielded primarily single allele-level genotypes. Excluding amplification failures and inability to obtain an NGS assignment (4% loci), 32 discrepant NGS allele assignments were attributed to poor PCR amplification or loss of reads during assembly by software. Complete exon sequences identified 53 known A alleles (48 common or well-documented (CWD)), 89 B alleles (83 CWD), 52 C alleles (47 CWD). Ten potential new alleles with exon variation outside of exons 2 + 3 and over 25% of samples with potential intron variation require further study. The ability to make assignments based on specific regions of the sequence (e.g., exons only) was critical so that typings could be rapidly transmitted to the registry prior to a more comprehensive analysis of the diversity. Conclusions The protocol for NGS of class I alleles provides accurate results and should facilitate high volume high resolution typing of donor registry populations. The extent of new variation in exons is limited but the potential for intron variation is high. C. Hurley: Other (Identify); Company/Organization; Intellectual property.