Very little is known about possible intra-tumoural genetic heterogeneity between primary tumours and lymph node metastases in head and neck squamous cell carcinoma (HNSCC). To investigate this phenomenon, we analysed 96 micro-dissected tumour samples for allelic imbalance at four of the most frequently altered chromosomal locations in HNSCC (3p14.2; 9p21; 11q23.3; 17p13.1) using microsatellite markers. From 23 patients, matched pairs of primary tumour and lymph node metastasis were analysed. Discordance in the allelic distribution was identified in 8 cases (35%). With one exception, the metastasis contained a more balanced allelic status than the primary tumour. In contrast, in a group of 25 tumours with two anatomically different samples from the primary tumour site, discordance was identified in only 3 tumours (13%). These results are compatible with the dissemination of subclones from the primary tumour site with a more balanced allelotype in the metastasis. In our opinion, several scenarios could explain this phenomenon. From a clinical point of view, genetic discordance between the metastasis and the primary tumour must be taken into consideration when establishing molecular biologic markers for choice of therapy and prognosis in head and neck cancer.