Human leukocyte antigen (HLA) expression is important for the elimination of tumor cells by the immune system and immunotherapy. Activated T cells directed against tumor-associated antigens are fully capable of recognizing and eradicating neoplastic cells. Therefore, HLA expression loss is considered to be a main factor in tumor development. We report for the first time HLA-A and HLA-B allele-specific expression analysis by immunohistochemical staining of fresh tumor tissue and 9 lymph node metastases of 15 patients with head-and-neck squamous cell carcinoma. Heterogeneous HLA expression and HLA expression loss was detected in 13 tumor patients. Approximately 50% of the tumors had allele-specific expression loss, which would have remained undetected using HLA monomorphic and locus-specific antibodies. In the majority of the patients with head-and-neck squamous cell carcinoma, HLA allele-specific expression loss differed between primary lesions and metastases. This is important for the efficacy of immunotherapy in these patients. It can be concluded that it is crucial to study HLA expression at the allele-specific level of primary lesions and metastases. It increases and refines our knowledge of HLA expression loss in tumorgenesis, which will improve the development of specific immunotherapy.