Collagen fibers are the main element of gingival connective tissue and contribute a leading role in the preservation of structural integrity and tissue function. Hence, its degradation is regarded as the main marker of periodontal disease progression.The aim of this study is to analyze and compare collagen fibers, their birefringence pattern in healthy and in diseased gingival tissues stained using picrosirius red stain (PRS) and the polarizing microscope.A total of 90 participants screened were divided into the control group (healthy gingiva) and experimental group (moderate periodontitis and severe periodontitis) based on the clinical parameters. Gingival tissue sections were stained with PRS and observed under the polarized microscope to assess the type of collagen fibers in healthy and diseased gingival tissue. Statistical analysis was performed using the one-way ANOVA and Tukey multiple comparison test.The healthy group revealed well-packed collagen in a parallel pattern with a strong birefringence, whereas in severe periodontitis group showed loosely packed collagen fibers in a haphazard pattern suggestive of severe destruction of the extracellular matrix. The moderate periodontitis group had a blended mixture of thick and thin fibers.Collagen fibers showed birefringence property when stained with PRS that helps in a better understanding of normal and pathological conditions.