Gene amplification, a common mechanism for oncogene activation in cancer, has been used as a tag for the identification of novel oncogenes. DNA amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC) and potential oncogenes have already been reported. We applied restriction landmark genome scanning (RLGS) to study gene amplifications and low-level copy number changes in HNSCC in order to locate previously uncharacterized regions with copy number gains in primary tumor samples. A total of 63 enhanced RLGS fragments, indicative of DNA copy number changes, including gains of single alleles, were scored. Enhanced sequences were identified from 33 different chromosomal regions including those previously reported (eg 3q26. 3 and 11q13. 3) as well as novel regions (eg 3q29, 8q13. 1, 8q22. 3, 9q32, 10q24. 32, 14q32. 32, 17q25. 1 and 20q13. 33). Furthermore, our data suggest that amplicons 11q13. 3 and 3q26. 3–q29 may be divided into possibly two and three independent amplicons, respectively, an observation supported by published microarray expression data.