Retinoblastoma gene (Rb1) is a tumor suppressor gene, which plays a pivotal role in cell cycle regulation, promoting G1/S arrest and growth restriction through inhibition of the E2F transcription factor. Abnormalities in the genes involved in cell cycle, including Rb1, have been reported in head and neck cancer (HNC) patients. Studies regarding Rb1 have been observed in different world populations but data is missing for Pakistani population. This study was aimed to analyze the genetic aberrations of Rb1 and their association with the development of HNC in Pakistani population. Genomic DNA was isolated from blood samples of 300 HNC patients and 270 controls. Salient coding region of gene was amplified by using Polymerase Chain Reaction (PCR). PCR conditions were optimized for each exon separately. Amplified products were analyzed for mutational screening using Single strand confirmation polymorphism (SSCP) technique followed by sequence analysis. Sequence analysis revealed five missense mutations g77082G>C, g77083G>A, g170220A>T, g170221G>C, g170228T>A, two frameshift mutations, two stop codon and two intronic substitutions in this study. The overall frequency of these mutations was 0.71. Frequency of nonsense mutations; Lys462stop (Novel) and Ser834stop (CM952105) were 0.15 and 0.14 respectively. We also report here novel missense mutations, frameshift mutation and a stop codon Lys462stop in HNC patients of Pakistani origin.This study suggests that the Rb1 germline mutations may contribute to genetic susceptibility for HNC. To our knowledge, this is the first report that Rb1 gene may be associated with risk of cancer in Pakistani population.