RB1, CCND1 and CDK4 are major cell cycle check point players related to head and neck cancer (HNC). Polymorphisms in these genes have frequently been reported in literature and are known to follow diverse patterns in relation to different populations. The current study was designed to screen these genes in head and neck cancer patients and control samples at DNA, RNA and protein level. Blood and tissue samples of pathologically confirmed head and neck cases were collected from different hospitals. Polymerase chain reaction (PCR) and single-strand confirmation polymorphism (SSCP) were used for germline screening, followed by sequence analysis. In second step Real time-PCR was performed to study expression of these genes at mRNA level. Immunohistochemistry was performed to evaluate the protein expression of these molecules in cancer tissues as well as controls. Epigenetic analysis was performed using methylation specific PCR. In the last part of the study, in vitro studies were carried out to characterize cancer cells with selected RB1 mutations and their response to drug resistance (doxorubicin). In first part of this study, PCR-SSCP and DNA sequencing were used to analyze the coding regions of RB1, CCND1 and CDK4 in 730 individuals (380 head and neck patients and 350 controls). Sequence analysis of coding regions of RB1 gene results in 18 mutations (one silent, 10 missense, 3 frame shift, 2 nonsense mutations and 2 splice site substitutions). Among these mutations, one silent synonymous mutation was observed in exon 12 (g70282A>G), 5 missense mutations observed in exon 14 (g76474C>T, g76475G>C, g76476A>G, g76467G>C g76468T>C and 2 missesnse mutations in exon 16 (g77041A>T, g77043A>G). One frameshift mutations in exon 12, was a result of deletion of five nucleotide GATGA (g70285_70289delGATGA). 2 frameshift mutations were found in exon 21, one novel frameshift mutation was due to the insertion of nucleotide G (g160601_160602insG) while the other was due to insertion of nucleotide A [(g160623_160624insA) (CI952130)]. In exon 14 and exon 24, 2 nonsense mutations Lys462stop (novel) and Ser834stop (CM952105) were also observed, respectively. Germline analysis of CCND1 coding revealed total 4 mutations, one missense, one frameshift, one silent and one in 3''UTR. One novel missense (g3578C>A) and one novel xframeshift mutation (g3383delA) was observed in exon 3. One silent variation as substitution of G>A (G870A [rs9344]) was observed in exon 4. In addition C>A transition (rs7177) was observed in untranslated region (3''UTR) of CCND1. CDK4 germline mutation analysis showed 2 missense mutations (g5051G>C and g5095G>C) in exon 2 and 2 missense mutations in exon 5 (g5906C>A and g5907C>G). One novel frameshift mutation as a result of three nucleotide deletion (g7321_23delTGA) was observed in exon 7. Additionally 2 novel frameshift mutations as a result of insertion of nucleotide G (g7121_7122insG) and deletion of nucleotide G (g7143delG) were also observed in 3''UTR. mRNA expression of Rb pathway genes (RB1, CCND1 and CDK4) was studied in 72 head and neck tumors samples and adjacent un-involved tissues (control), using real-time PCR. A statistically significant (p<0.001) down-regulation of RB1 was observed in tumor samples compared to control samples. CCND1 (p<0.004) and CDK4 (p<0.02) showed over expression in tumor samples versus control samples. Immunohistochemical analysis of RB1, CCND1 and CDK4 suggested 67% tumors exhibited down-regulated expression of RB1, 62% of tumors showed up-regulated CCND1 expression and 54% of tumors showed up-regulated expression of CDK4. Epigenetic analysis was performed to check methylation status of RB1 promoter region, through methylation specific PCR (MSP). Methylation analysis of RB1 promoter region after sequence analysis revealed that 3% of CpG island was methylated in patients as compared to controls (un-involved tissues). In vitro characterization of selected RB1 mutations was carried out using cancer cell lines, revealed that RB1 mutations in important functional domains result in abnormally reduced protein stability causing defects in RB1 functioning which may play a role in tumor proliferation. Results indicate that loss of RB1 function makes the condition selectively advantageous in tumor aggression which highlights the importance of RB1 in tumor protection. Results from this study also suggest that cells expressing mutant RB1Arg455Ser-V5 and RB1Ile835Asn-V5 may get a survival advantage against doxorubicin induced cytotoxicity, which may enhance drug resistance.