Oncogene-expressing human papillomavirus type 16 (HPV16) is found in a subset of head and neck squamous cell carcinomas (HNSCC). HPV16 drives carcinogenesis by inactivating p53 and pRb with the viral oncoproteins E6 and E7, paralleled by a low level of mutations in TP53 and allelic loss at 3p, 9p, and 17p, genetic changes frequently found in HNSCCs of nonviral etiology. We hypothesize that two pathways to HNSCC exist: one determined by HPV16 and the other by environmental carcinogens. To define the critical genetic events in these two pathways, we now present a detailed genome analysis of HNSCC with and without HPV16 involvement by employing high-resolution microarray comparative genomic hybridization. Four regions showed alterations in HPV-negative tumors that were absent in HPV-positive tumors: losses at 3p11. 2–26.3, 5q11. 2–35.2, and 9p21. 1–24, and gains/amplifications at 11q12. 1–13.4. Also, HPV16-negative tumors demonstrated loss at 18q12. 1–23, in contrast to gain in HPV16-positive tumors. Seven regions were altered at high frequency (> 33%) in both groups: gains at 3q22. 2-qter, 5p15. 2-pter, 8p11. 2-qter, 9q22–34.1, and 20p–20q, and losses at 11q14. 1-qter and 13q11–33. These data show that HNSCC arising by environmental carcinogens are characterized by genetic alterations that differ from those observed in HPV16-induced HNSCC, and most likely occur early in carcinogenesis. A number of genetic changes are shared in both tumor groups and can be considered crucial in the later stages of HNSCC progression.