AIMS: It is of interest whether expression of potentially predictive biomarkers changes during chemotherapy, for accurate evaluation after first-line chemotherapy. This study aimed to evaluate changes in RRM1 expression during chemotherapy. MATERIALS AND METHODS: RRM1 immunohistochemistry was performed on tumour samples from a total of 118 NSCLC patients with stage T1-4N0-2M0 disease. Samples were included from 65 patients treated with paclitaxel and carboplatin before surgery [neoadjuvant chemotherapy (NAC) group], and 53 patients who had undergone surgery but not chemotherapy [operation (OP) group]. RESULTS: Discordant RRM1 expression (low versus high) was observed in 32% and 43% of paired diagnostic and subsequent resection specimens in the OP group and NAC group, respectively (P = 0.913). Ten (33%) and 12 (23%) tumours in the NAC group and the OP group, respectively, had increased RRM1 expression in the resection specimens (P = 0.289), and 12 (40%) and 19 (36%) tumours had decreased expression (P = 0.707). Eleven (50%) lymph node metastases had higher RRM1 expression following chemotherapy, and two (7%) had decreased expression. CONCLUSIONS: The substantial discordance between paired samples emphasizes the need for sufficient tumour tissue in biopsies when RRM1 expression is evaluated. No change in RRM1 expression was observed in primary tumours, but expression seemed to be higher in N2 lymph node metastases following chemotherapy. Tumour heterogeneity and potential post-chemotherapy changes should be considered when RRM1 expression is evaluated.