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Aim Sanger sequencing of HLA suffers from the inability to set phase in certain heterozygous combinations. NGS overcomes these problems, but the reliability is not well described. We tested the NGS TruSight® HLA Sequencing Panel (Illumina) for its ability to provide full length, genomic, accurate, unambiguous, phase-resolved HLA genotyping in a single assay on a panel of 145 specimens. Residual clinical, PT, and IHWG DNA samples from blood, buccal swabs, cell lines of varying quality, concentration, and age, were selected to cover every known antigen, as well as rare and novel alleles. Methods The 145 samples (2070 alleles) included 26 novel variants, 7 null alleles, and 7 PT samples. We used the Illumina NGS HLA genotyping system end-to-end, from Long-Range PCR and library preparation to sequencing on the MiSeq. FASTQ files were analyzed by Conexio Assign, providing phase-resolved genotyping results for HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, -DPB1. We calculated concordance and unambiguous allele level identity in comparison to previously typed high resolution typed results (Sanger/SSP/SSO combined). Results 87.1% of all clusters (500 cycles) had a Q30 quality score (error rate of 0.1–0.01%). Concordance with original typing was 98.5%. Subdividing concordance, we found: 65.1% identical and unambiguous in reference and NGS; 20.8% unambiguous NGS, ambiguous reference; 9% unambiguous reference, ambiguous NGS; 3.7% ambiguous in both reference and NGS; and 1.5% discordant. Unambiguous reference/ambiguous NGS was the result of primer placement. Discordance (31 alleles) was due to novel alleles, reference errors, homopolymers and microsatellites, pseudogenes, and 3 instances of contamination. Benefits of NGS included discovery and resolution of novel alleles, identification and correction of IHWG reference and clinical mistypings. Analysis of 24 samples for 11 loci took ∼3–3.5 h. Conclusions TruSight® HLA Sequencing system is a reliable, accurate, comprehensive, ultra-high-resolution HLA typing method that can be easily implemented in the laboratory. For labs needing the highest resolution typing, it reduces the number of ancillary tests that must be performed to resolve ambiguities from ∼50% down to ∼14%. For registry labs, the concordance rate equals or exceeds 98.5%. F. Yamamoto: Grant/Research Support; Company/Organization; Illumina, Conexio. A. Lindell: Grant/Research Support; Company/Organization; Illumina, Conexio. B. Baas: Grant/Research Support; Company/Organization; Illumina, Conexio. A. Crawford: Grant/Research Support; Company/Organization; Illumina, Conexio. M. Won: Grant/Research Support; Company/Organization; Illumina, Conexio. N. Baird: Grant/Research Support; Company/Organization; Illumina, Conexio. M.W. Anderson: Grant/Research Support; Company/Organization; Illumina, Conexio. J. Nytes: Grant/Research Support; Company/Organization; Illumina, Conexio. J.J. Schiller: Grant/Research Support; Company/Organization; Illumina, Conexio. D. Goodridge: Grant/Research Support; Company/Organization; Illumina, Conexio. D.B. Tyan: Grant/Research Support; Company/Organization; Illumina, Conexio. [ABSTRACT FROM AUTHOR]