Epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9) expression in resected non-small cell lung cancer (NSCLC) has been associated with a poor prognosis. Specific inhibitors have been developed to these molecules and have entered into clinical practice. We performed immunohistochemical staining on a series of 36 resected cases of NSCLC in parallel with the associated preoperative diagnostic biopsies in order to determine whether expression of these markers in the tumour could reliably be predicted from the result obtained with the small diagnostic biopsy. The results demonstrated considerable intratumour heterogeneity of expression for both markers in the resected tumours, and this was associated with a poor negative predictive value for the result obtained with the diagnostic biopsy. We conclude that, staining small diagnostic biopsies for EGFR and MMP9 is unlikely to be helpful in defining tumour status for these markers and allowing targeted therapy.