Aims: To compare the gene expression profiles and proliferation rates of fibroblasts from the oral lining and masticatory mucosae.Materials and methods: Primary human fibroblasts were retrieved from the posterior masticatory hard palate and the lining alveolar mucosa of five individuals. The gene expression profile was evaluated using total RNA sequencing. The proliferation rate was determined colorimetrically.Results: Substantial differences in specific gene groups and pathways were observed between fibroblasts from the two tissues. Significantly enriched gene ontology processes were focused on the extracellular components. Lining mucosa fibroblasts exhibited significantly higher expression of the principal structural collagens, cranial neural crest markers, and homeobox genes associated with positional memory. Masticatory mucosa fibroblasts showed greater expression of genes related to transforming growth factor-β signalling, which may be associated with fibrosis. In addition, they expressed higher levels of the EP 2 prostaglandin E 2 receptor and Toll-like receptor 1. Finally, masticatory mucosa fibroblasts exhibited a 10%-30% higher proliferation rate.Conclusions: Fibroblasts from the lining and masticatory oral mucosae are phenotypically heterogeneous, presenting distinct gene expression profiles and proliferation rates. These features may contribute to their specific physiological functions and have relevance for potential therapeutic applications.