Objective: Breast cancer comprise approximately one third of malignant cases in women and is considered as the most common invasive condition in women at an age range of 15-54 years and as the second most prevalent cause of mortality at an age range of 55-74 years. Tumor inhibiting factor p53 is a vital homeostatic regulator and its inactivation at the related gene or molecule could lead to tumor growth and development in various tissues. Therefore, in this research study it was tried to evaluate the diagnostic methods Ag-NOR and p53 immunohistochemistry in malignancy of mammary gland using cytochemical staining methods and its relationship with tumor grade. Materials and Methods: In this research study, 50 referred breast specimens to Deaprtment of Pathology (Sina Hospital, Hamedan) were studied. They were processed as usual and 3 micrometer sections were prepared from related blocks. Then, staining methods for nucleolus organizing regions (Ag-NOR) and p53 immunohistochemistry were applied. Out of these specimens, 41 had malignancy (40 cases of invasive ductal carcinoma and 1 case of invasive lobular carcinoma) and 9 cases were normal. The latter cases were compared with malignant ones. Tumor grade in studied individuals was I (3 cases; 7.3%), II (23 cases; 56.1%), and III (15 cases; 36.6%) respectively. Results: Statistical analysis of data showed that there is only a significant difference regarding frequency distribution of cluster shape and there is no such difference for satellite shape, satellite size, and cluster size. In addition, staining intensity for p53+, p53++, and p53+++did not show any significant difference in various grades of the disease. Using Spearman regression analysis, it was found out that there was a relationship between p53 negative and p53+ (r = 0.723) (p<0.01) and between p53+ and p53++ (r = 0.78). Furthermore, it was found out that a higher expression of p53 protein is negatively correlated with darklystained granules using silver nitrate method. Conclusion: These findings showed that higher expression of p53 protein is negatively correlated with darkly-stained granules using Ag-NOR method and this may indicate its antitumor activity. It appears that this method is an essential tool for evaluation of normal and malignant cases of breast tissue regarding its replication pattern and intensity and expression of those chromosomal segments which are involved in encoding of ribosomal RNA.