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We investigated intratumoral genetic variability using 13 of the most frequently altered microsatellite loci in primary head and neck squamous carcinoma to determine the extent and the implication of this feature on the interpretation of the genetic analysis of these tumors. DNA extracted from four spatially different samples of 17 prospectively resected specimens, in addition to matching normal mucosa, was tested. Overall, the highest incidence of loss of heterozygosity was noted at chromosomes 3p and 9p loci (70% in each). Chromosomes 8p and 17p loci showed 35% and 40% loss of heterozygosity, respectively. Loss of heterozygosity was homogeneously manifested in all different samples of tumors with these alterations. Microsatellite instability was noted in two (17%) tumors, one of which showed intratumoral heterogeneity. Our study indicates that single sample analysis may reflect the genetic alterations in a given primary head and neck squamous carcinoma using these markers.