p21 is a cyclin dependent kinase inhibitor, that plays an important role in cell growth, differentiation and apoptosis. The aim of present study was to analyze the mutation and expression of tumor suppressor gene (p21) in canine tumors. A total of 26 tumors (mammary tumors and mast cell tumors) and normal samples from different breed of dogs were analyzed by using direct DNA sequencing and quantitative RT-PCR for mutation and expression profiling of p21gene respectively. The coding exon of p21 gene (Exon2 and 3) did not show any polymorphism in studied samples while 69% tumors showed up-regulation of gene. Seventy five percent mammary tumors showed up-regulation of gene with highest fold change 8.69, 7.6, 5.63, 5.21 and 60% mast cell tumor showed up-regulation of gene with highest fold change 7.31. These results indicate that altered expression of tumor suppressor gene may be involved in malignant progression of tumors and might be helpful in the diagnosis and prognosis of canine tumors.