Background: Oral cancer is a major problem globally and more so in Pakistan as it ranks as the second most common malignancy with an aggressive nature and a high mortality despite modern, advanced treatment options. The strong causal association with smoked and chewed tobacco and its substitutes, prevalent in this region makes it imperative to consider the mutation analysis of molecular genetic profile and the role of HPV in oral oncogenesis. It would help in early and accurate detection of targets for therapeutic implementations. Objectives: To identify the genetic mutations in a subset of Pakistani patients of oral cancer and precancer with dominant exposure to a different set of environmental carcinogens as betel quid, arecanut and its substitutes. Moreover to identify the role of HPV in oral carcinogenesis with interactive relationship of chemical and viral carcinogens. Methodology: Hundred clinically diagnosed and histologically confirmed cases of OSCC and 50 cases of oral premalignant lesions were included in the study. Details of demographic data alongwith personal habits concerning tobacco related carcinogen exposure were noted. A meticulous local and general examination was conducted. Controls were included for histological and molecular comparisons. The tissues obtained at biopsy or surgical resections were subjected for routine histopathological reporting followed by Immunohistochemical analysis of commonly reported mutated oral cancer genes in oral cancer viz p53, p16, H ras, CyclinD1, C Myc, and EGFR. HPV 16 and18 status was detected by Q- PCR. Statistical evaluation was done by SPSS version 16. Results: The ages of 100 patients of OSCC ranged from 25-80 years and 50 preneoplasia from 26 – 65 years with the mean age being 47.84+/- 12.18 and 40.22+/- 9.66 respectively. In OSCC group 74 were males and 26 were females, in preneoplasia 35 were males and 15 females; the male to female ratio being 2.84:1 and 2.33:1, respectively. Ninety one patients of OSCC and 46 (92%) of oral preneoplasia were exposed to tobacco, BQ and BQS, 29 and 32 were exposed to a combination of these. Cheek was the most common site for OSCC (50%) and OPL (42%). Nearly half (48%) of OSCC were well differentiated and majority (74%) presented in advanced stages III and IV. In oral preneoplasia cases 80% presented as leukoplakia and 50% showed mild dysplasia. HR- HPV 16 and 18 were found to be positive in 15 (15%) cases of OSCC and 3 (6%) cases of oral preneoplasia presented with greater prevalence of HPV 16. p53 nuclear protein positivity was seen in 70 (70 %) cases of OSCC and 27 (54 %) of OPL significant correlation (p <0.05) with chemical risk factors was found. Lack of immunoexpression of p16 was observed in 82 (82 %) cases of OSCC and 43(86%) of OPL. EGFR revealed membranous staining in 68 ( 68 %) of cancer cases and in 23 (46 %) dysplastic oral lesions. A strong association was seen with the most common BQ and tobacco chewing habit and significant correlation was observed with stage of oral cancers. H ras cytoplasmic immunoexpression was detectable in a significantly high proportion (50%) of oral cancers and premalignant lesions 17 (34 %). C myc nuclear positivity was observed in 36 /100 (36%) cases of Invasive oral squamous carcinomas and 11 / 50 (22%) cases of premalignant dysplasias. Cyclin D1 gene overexpression with evidence of nuclear positivity was detected in biopsy materials of 40 (40%) oral cancers and 14 (28%) dysplastic oral precancerous lesions. Highly significant coexpression (p<0.05) of HPV was observed with p16 and negatively significant with p53. A significant negative association (p < 0.05) was seen among p16 positive oral cancer cases and p53 and EGFR. Conclusion: A strong etiologic role of chemical carcinogens as smoked and chewed tobacco, betel quid and its substitutes is confirmed in Pakistani population. A frequency of molecular alterations was detected in p53, p16, EGFR and H ras; in early stages of oncogenesis. HR- HPV was detected in a substantial number of oral cancers and preneoplasias but chemical carcinogens have a dominant role. Overexpression of p16 was found in HR HPV positive cases. Key words: Oral Squamous cell carcinoma, Oral premalignant lesions, Betel quid, tobacco, areca nut, immunohistochemistry, molecular markers, HPV, PCR.