Background: Little is known about the molecular genetic events that contribute to the pathogenesis of squamous cell carcinoma of the upper aerodigestive tract. Previous molecular genetic studies have been limited to the identification of mutations of the p53 (also known as TP53) tumor suppressor gene, activation of a limited set of oncogenes, allelic loss at 3p and other locations, and occasional association with human papillomavirus infection. Purpose : Our purpose was to screen tumor tissue and blood from patients with squamous cell carcinoma of the upper aerodigestive tract for loss of heterozygosity at polymorphic loci corresponding to each of the autosomal chromosomes and to identify the locations of additional putative tumor suppressor genes, other than RB (also known as RB1) and p53, that may contribute to the pathogenesis of this disease. Methods: Tumor tissue and blood were obtained from 68 consecutive patients with squamous cell carcinoma of the upper aerodigestive tract. In all cases, tumor tissue was obtained from the center of the surgical specimen. The relative absence of non-neoplastic tissue was confirmed by frozen-section histologic examination of immediately adjacent tissue. Initially, 30 paired tissue and blood samples were tested for loss of heterozygosity by polymerase chain reaction (PCR) to amplify 43 different highly polymorphic sequences containing small oligonucleotide repeats. After PCR amplification, with unique oligonucleotides flanking the repeat, visualization and sizing of the alleles on DNA sequencing gels were performed. Specific loss of heterozygosity was distinguished from random genetic loss due to generalized chromosomal instability if it occurred in more than 20% of specimens tested for a particular marker. Results : Significant loss of heterozygosity (>20%) occurred at alleles at chromosome bands 3p21 (32%), 3p25-26 (56%), 8pter-21.1 (31%), 13q14 (27%), and 17p12 (45%). Loss of heterozygosity at more than two loci was significant with a poor prognosis ( P = .039). Conclusions: These findings demonstrate that squamous cell carcinoma of the upper aerodigestive tract exhibits genetic alterations at multiple loci and that allelic loss at more than two locations is indicative of a poor prognosis (the likelihood of the patient dying of disease). Implications : While tumor suppressor genes at 3p (VHL), 13q (RB), and 17p (p53) have been identified, altered genes at other loci on and on 8p have not yet been characterized. Furthermore, the genotype at these loci for squamous cell carcinoma of the upper aerodigestive tract has prognostic importance and may identify the patients who should receive the most aggressive treatment.