Squamous cell carcinomas arising from various subsites within the head and neck (HNSCC), while histologically identical, have substantial differences in survival and recurrence rates. Controversy exists as to whether this reflects physical differences between subsites or fundamental molecular heterogeneity. In this study, we used two proteomic approaches to evaluate HNSCCs for differences in protein expression between oral cavity, oropharynx, larynx and hypopharynx subsites. A tissue microarray (TMA) was constructed consisting of 71 patients with HNSCC. This TMA was queried for expression of 4 cell-cycle and regulatory proteins chosen a priori for their known roles in cancer, using automated quantitative analysis (AQUA) of protein expression. Frozen tissue samples from 14 patients with histologically confirmed HNSCC were enriched for tumor and normal tissue by laser capture microdissection. Total protein was extracted, analyzed by 2D-difference gel electrophoresis (2D-DIGE) with saturation dye labeling, and evaluated for differential protein expression between subsites. AQUA analysis likewise revealed no difference between subsite for cyclin D1, p53, Rb, or p14 expression. Proteomic analysis was based on 28 gels (14 cancer, 14 adjacent normal) and 732 spots were identified as matching across >90% of gels. Statistical analysis detected no significant differences in protein expression between subsites. Observed differences in outcomes between HNSCCs from different subsites are unlikely to reflect differences in tumor biology between subsites. It is possible that the observed heterogeneity in clinical behavior among HNSCCs may be based on fundamental differences in carcinogenesis such as viral versus chemical carcinogenic insult.