Introduction: Breast cancer (BC) is the most common cancer in women, which is the second most common malignancy in terms of mortality and prevalence after lung cancer. BC is a group of very diverse diseases that can be detected at a molecular, histopathological and clinical level. Objectives: The aim of the present study was to evaluate the expression of CK5/6 and its relationship to some of the factors affecting prognosis, such as tumor grade, lymph node involvement. Patients and Methods: In this cross-sectional study with descriptive and analytical aspects, 40 malignant and 20 benign tumors were collected in 2020 from the archives of the pathology department of two educational hospitals in the city of Ahvaz. The demographic and clinical characteristics of each specimen, including age, patient, lymphovascular and perineural invasion, lymph node involvement, tumor grade and tumor type were extracted from the patients’ record. Then the expression of CK5/6 intensity staining was examined by immunohistochemistry. Results: All benign breast lesions had positive expression for CK5/6 and staining intensity between six and nine. In the malignant group, 33 samples showed negative expression of CK5/6 and only seven samples (17.5%) showed positive expression with a low-staining index. A positive expression with a low-staining index. All seven positive specimens were invasive ductal carcinoma (IDC) lesions with staining index 2, 4, and 6. Additionally, none of the ductal carcinoma in situ (DCIS) specimens which immunostained were positive for CK5/6. In the present study, all IDCs with weak expression of CK5/6 were grade III. No statistically significant relationship was observed between perineural and lymphovascular invasion and lymph node involvement with the intensity of CK5/6 expression. Conclusion: Our study showed that cytokeratin immunohistochemical intensity is able to distinguish benign lesions from malignant IDC and DCIS lesions and accordingly in determining of tumor grade after weak staining in high-grade IDC, which may be due to squamous metaplasia in these tumors; however, more extensive research with a larger sample size are required to assess its effect in lymphovascular and perineural invasion and also lymph node involvement.