The major risk factor for oral squamous cell carcinoma (OSCC) in developing countries like Pakistan is the excessive chewing habit of paan, chhaliya, tobacco, niswar, gutka and manpuri. Mutagens can damage DNA and generate promutagenic lesions. It has been reported that exon 4-9 were the hot spots of the mutation in the tumor suppressor gene. This study aims to find out the loss of TP53 functions due to mutation/polymorphism caused by genomic alteration and interaction with tobacco and its related ingredients in Pakistan. A total of 260 OSCC patient’s tissue and blood specimens were collected with informed consent from local hospitals of Karachi. Mutations in exons 2-11 of p53 gene were examined by polymerase chain reaction and single stranded conformational polymorphisms (PCR-SSCP) and directly sequenced. The current study revealed a novel mutation in exon 7 of p53 gene. This mutation was observed only in the tumors of the OSCC patients. The “AGT” to “ACT” missense mutation was identified at position 719 at TP53. This change substitutes the amino acid serine with threonine at position 240 of p53 protein suggesting that the threonine allele of p53 gene is associated with mutation in conserved region. This novel missence mutations in DNA binding domain indicated that the DNA structure may be damaged by the use of exogeneous DNA-damaging agents including tobacco related carcinogens present in gutka, niswar and manpuri, which may result in the loss of cellular processes and p53 protein function.