Background: Oral squamous cell carcinoma (OSCC) encompasses all malignancies originating in the oral cavity and globally it is the most common cancer. It is a fact that tumor stroma plays a vital role in tumor progression as it contains significant stromal glycoprotein and collagen. Therefore analysis of collagen fibers is of immense value which in turn aids in predicting the biological behavior of the tumor. Aim: The aim of the present study was to evaluate birefringence pattern of collagen fibers and to determine varying proportions of stromal collagen in different grades of OSCC using picrosirius red and polarizing microscopy. Materials and Methods: A total of 28 cases, which included 22 histopathologically diagnosed cases of OSCC, of which 8 were of well differentiated, 8 moderately differentiated and 6 poorly differentiated OSCC. 6 cases of control group were also retrieved. The sections were stained with both Haematoxylin and Eosin (H&E) and picrosirius red and they were examined under polarizing microscope. Statistical analysis: The statistical differences were analyzed by one way ANOVA. Result: It was observed that thick collagen fibers decrease and thin collagen fibers increase with dedifferentiation of OSCC and also polarization colors of thick and thin fibers changes from reddish orange (RO) to yellowish orange (YO) to greenish yellow (GY) from well to moderate to poor grades of OSCC respectively. Conclusion: Thus the present study suggested that there was a change in polarization colors of thick fibers from RO to GY, which could be due to change in mature form of collagen into immature form as the tumor progress from well to poorly differentiated OSCC. The significance of this study lies in birefringence of stromal collagen fibers observed around tumor islands in different grades of OSCC.