Despite adequate surgical resection, oral squamous cell carcinoma (OSCC) shows a high rate of recurrence and metastasis, which could be explained by the presence of molecular alterations in seemingly normal tumour margins and the entire oral mucosa. The aims of this study were (1) to assess the presence of gene amplification (c-Myc and HER2) and promoter methylation (p14 and p16) in the tumours, tumour margins, and unaffected oral mucosa of 40 OSCC patients, and (2) to evaluate the possibility of using these alterations as prognostic markers. c-Myc and HER2 genes were quantified by means of real-time PCR (qPCR), and p14 and p16 methylation status was determined by methylation-specific PCR (MSP PCR). All tissues examined exhibited molecular alterations in various proportions. Tumour tissues, as expected, showed the highest prevalence of alterations, while oral mucosa showed the lowest. Multiple alterations (co-alterations) in tumours and tumour margins were significantly more frequent than in unaffected oral mucosa (P<0.001 and P=0.027, respectively). HER2 amplification in margin tissue (P<0.001) and swabs (P=0.013), as well as the existence of three co-alterations in margins (P=0.001) and macroscopically unaffected oral mucosa (P<0.001) were correlated with shorter disease-specific survival.