The nature and frequency of human histocompatibility leukocyte antigen (HLA) class I loss mechanisms in primary cancers are largely unknown. We used flow cytometry and molecular analyses to concurrently assess allele-specific HLA phenotypes and genotypes in subpopulations from 30 freshly isolated cervical tumor cell suspensions.Tumor-associated HLA class I alterations were present in 90% of the lesions tested, comprising four altered pheno/genotype categories: (a) HLA-A or -B allelic loss (17%), mostly associated with gene mutations; (b) HLA haplotype loss, associated with loss of heterozygosity at 6p (50%). This category included cases with additional loss of a (third) HLA-A or -B allele due to mutation, as well as one case with an HLA class I-negative tumor cell subpopulation, caused by a beta2-microglobulin gene mutation; (c) Total HLA class I antigen loss and retention of heterozygosity (ROH) at 6p (10%); and (d) B locus or HLA-A/B downregulation associated with ROH and/or allelic imbalance at 6p (10%). Normal HLA phenotypes and ROH at 6p were observed in 10% of the cases. One case could not be classified (3%). Altered HLA class I antigen expression occurs in most cervical cancers, is diverse, and is mainly caused by genetic changes. Combined with widespread tumor heterogeneity, these changes have profound implications for natural immunity and T cell-based immunotherapy in cervical cancer.