Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3′ untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.