Forensic dentistry is a branch of dentistry which in collaboration with legal profession serves an important role to maintain justice system of a country. Forensic dentists play a major role in identification of an individual. Within the literature various methods have been found to be useful in gender differentiation. An attempt was made for differentiation of gender using picrosirius red and polarization microscopy. To evaluate picrosirius red and polarization microscopy as a tool for gender differentiation by observing birefringence pattern and distribution of thick and thin collagen fibers in males and females. Labial mucosal tissue obtained from 30 deceased individuals (18 male and 12 female) during autopsy was fixed in 10% formalin at 12th hour. Tissue was processed, sectioned and stained using picrosirius red stain and the birefringence pattern of collagen fibers were studied with polarization microscope. The results were statistically analyzed using Z-test and one-way ANOVA to draw the significance. The proportion of thick and thin fibres among males and females were compared. It was found that there was statistically significant difference in proportion of thick and thin fibers between male and female. Thick fibres in males were (78.13%) more than females (65.74%) and thin fibres were more in females (34.24%) than males (21.32%). Picrosirius red and polarization microscopy may be used as a tool for gender differentiation. Yet the manner of death has to be considered during gender differentiation using this method, as in the present study out of 30 cases studied three cases of death due to debilitating diseases and poison consumption showed altered collagen distribution.