Several risk factors associated with colorectal cancer (CRC) have been identified including β-catenin/CTNNB1 hotspot mutations. The levels of β-catenin within a cell are regulated via phosphorylation of the N terminus of β-catenin by GSK-3β. Thus far three serines (S33, 37, 45) and one threonine (T41) are considered to be the substrates for GSK-3β phosphorylation. In the present investigation an attempt was made to study the role of β-catenin mutations in exon-3 in 60 colorectal cancer patients from Kingdom of Saudi Arabia (KSA). The hot spot mutation region of β-catenin exon 3 was evaluated in matched tumor and normal tissues using PCR and direct sequencing. Sequencing of exon 3 of the CTNNB1 gene revealed an activating mutation (S33F) in one of the tumor samples as compared to the normal tissue from the same patient where there was no such mutation found. Immunohistochemical staining showed the accumulation of β-catenin protein both in cytoplasm and in the nuclei of cancer cells as compared to normal tissue.