Classical Hodgkin lymphoma (cHL) is characterized by sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells amid reactive host background, complicating the acquisition of neoplastic DNA without extensive background contamination. We overcame this limitation by using flow-sorted HRS and intratumor T cells and optimized low-input exome sequencing of 10 patient samples to reveal alterations in genes involved in antigen presentation, chromosome integrity, transcriptional regulation, and ubiquitination. beta-2-microglobulin (B2M) is the most commonly altered gene in HRS cells, with 7 of 10 cases having inactivating mutations that lead to loss of major histocompatibility complex class I (MHC-I) expression. Enforced wild-type B2M expression in a cHL cell line restored MHC-I expression. In an extended cohort of 145 patients, the absence of B2M protein in the HRS cells was associated with lower stage of disease, younger age at diagnosis, and better overall and progression-free survival. B2M-deficient cases encompassed most of the nodular sclerosis subtype cases and only a minority of mixed cellularity cases, suggesting that B2M deficiency determines the tumor microenvironment and may define a major subset of cHL that has more uniform clinical and morphologic features. In addition, we report previously unknown genetic alterations that may render selected patients sensitive to specific targeted therapies.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Weill Cornell Medical College, Department of Pathology and Laboratory MedicineNorthwestern University, Department of Pathology, Feinberg School of MedicineRush University Medical Center, John H. Stroger Jr Hospital of Cook CountyWeill Cornell Medical College, Department of PediatricsHumanitas Clinical and Research Center, Humanitas Cancer CenterUniversity of Milan, School of MedicineWeill Cornell Medical College, Institute for Computational BiomedicineUniversidade Estadual Paulista, Departamento de Clínica Médica, Faculdade de Medicina de Botucatu