Head and neck squamous cell carcinoma (HNSCC) is a diverse group of cancers that are frequently aggressive in their biologic behavior. Inactivation of tumor suppressor gene (TSG) is one of the most critical steps leading to HNSCC. Loss of heterozygosity analysis is very sensitive method for the detection of frequent allelic loss in a chromosomal locus. This method has been considered as an important evidence for the localization of TSGs. We analyzed loss of heterozygosity (LOH) at chromosome 4q22-35 region by using 14 polymorphic microsatellite markers in 83 matched normal and HNSCC tissues. LOH was detected at least in one location in 71 of 83 (86%) tumor tissues. Frequent deletions were detected at the location of microsatellite markers, D4S2909 (46%), D4S2623 (51%), D4S406 (48%), D4S1644 (45%) and D4S2979 (40%). Four different frequently deleted regions at 4q22, 4q25, 4q31 and 4q34-35 were observed. These regions include several putative TSGs such as Caspase-6, SMARCAD1, SMARCA5, SAP30 and ING2. Further molecular analysis of each gene should be performed to clarify their roles in head and neck squamous cell carcinogenesis.