Purpose: Recent evidence suggests that small subpopulations of stem-like cells are accountable for tumour initiation, progression and metastasis. Until now, studies were focused exclusively on the characterization of these cell populations within the tumour itself, while tumour margins were neglected, although it is known that the histological and molecular status of tumour margins may play a significant role in the course of the disease. Therefore, the aims of the study were to isolate cells from oral squamous cell carcinomas and their respective margins, to characterize these cells using specific markers, to assess their self-renewal potential and determine their chemoresistance. Methods: Cell cultures were obtained from 12 tissue specimens (6 tumours and 6 margins). Total RNA was extracted and gene expression analysis was done by real-time PCR (RTPCR). Flow cytometry, immunocytometry, sphere formation and MTT assays were also applied. Results: With minor differences, cells originating from both tumours and tumour margins showed the presence of stem cell markers CD133, Nanog, Sox2, CD44, and Oct4, had the capacity to form spheroids and showed chemoresistance. Conclusions: Subpopulations of margin cells appeared to have sternness properties which might raise the question of re-evaluation of optimal surgical management