Background and Aim: Fibrous dysplasia (FD) and ossifying fibroma (OF) are the most important fibro-osseous lesions (FOLs) of the jaws with similarities in radiographic and morphological features while showing completely different biological behavior. Limited studies have been evaluated immunohistochemistry markers, such as osteopontin (OPN) and osteocalcin to help differentiate these two lesions. This study aimed to assess the immunoexpression of OPN and Ki67 as potential markers for differentiation of different FOLs. Materials and Methods: This cross-sectional study was conducted on 12 FD, 19 OF, and eight FOL samples retrieved from the archives of the department of oral pathology. The specimens were examined immunohistochemically using streptavidin-biotin method for OPN and Ki67. The intensity score (IS), proportional score (PS) and total score (TS) were assessed in hard and soft tissue matrix and in mesenchymal cells for Ki67. The data were analyzed by independent samples Kruskal-Wallis. Results: Osteopontin showed positive immunoreaction in both stromal and trabecular components of all FDs and OFs. Among the scores, PS and TS of bone trabeculae were significantly different in FD and OF (P=0.005). Nevertheless, no significant difference was observed in Ki67 expression in mesenchymal cells (P=0.880) and OPN scores in soft tissue matrix between the lesions; their P-value were 0.336, 0.340 and 0.415 for IS, TS, and PS, respectively. Conclusion: Osteopontin can serve as a useful marker for differential diagnosis of FD and OF. However, we suggest evaluation of other NCMPs, especially functionally similar molecules such as bone sialoproteins (BSPs) in FOLs for differential diagnosis.