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Aim To treat mucogingival problems palatal masticatory mucosa is used as a donor material. This study aimed to determine the thickness of donor palatal mucosa and associations of age and gender in Indian subjects aged 14 to 60 years by direct clinical technique. Materials and methods Forty systemically and periodontally healthy Indian subjects (20 males; 20 females; age range of 14 to 60 years) enrolled in this study. Under local anesthesia bone sounding technique was performed at 15 defined and predetermined points. The Wilcoxon test was used to determine the difference in mucosal thickness between the age groups and between genders. Significance of the difference between individuals of different body mass index (BMI) was assessed by independent t-test. Results With mean thickness of 2.0 to 3.7 mm, the younger age group demonstrated significantly thinner donor mucosa (mean 2.8 ± 0.3 mm) than the older age group (mean 3.1 ± 0.3 mm). Within the same age group though statistically not significant; males had thicker mucosa than females. From the canine to second premolar areas and in the sites furthest from the gingival margin mucosa thickness increased. When correlated with subject's BMI, in all probed sites males demonstrated significantly a thicker mucosa than females. Within each group subjects with high BMI demonstrated thicker donor mucosa. Conclusion Current study showed that in both adult and young individuals, premolar and canine areas can be the right site for harvesting donor tissue. Even in young subjects subepithelial connective tissue (CT) graft can be harvested in adequate volume. Donor tissue thickness can be influenced by factors like genetics and race, which need to be further evaluated. Clinical significance Premolar and canine areas serve as appropriate choice to harvest palatal masticatory mucosa. Even in young patients the volume of donor tissue available is sufficient enough to consider the subepithelial CT graft procedure. Donor tissue thickness can be influenced by factors like genetics and race, which need to be further evaluated. How to cite this article Anuradha BR, Shankar BS, John B, Prasad KARV, Gopinadh A, Devi KNN. Assessment of Palatal Masticatory Mucosa: A Cross-sectional Study. J Contemp Dent Pract 2013;14(3):536-543.