Simple SummaryHER2 intratumoral heterogeneity (ITH) is a well-known phenomenon in breast cancer, defined as the coexistence of subpopulations of tumor cells with different HER2 gene or protein expression within a tumor. HER2 ITH has been reported in up to 40% of breast cancers and to be associated with poor prognosis in patients with anti-HER2 targeted therapies and was proposed to be a potential mechanism for anti-HER2 resistance. HER2 ITH can be divided into non-genetic and genetic ITH based on different HER2 genetic amplification and genetic ITH has clustered, mosaic and scattered distribution patterns. HER2 ITH interpretation can be challenging, but digital image analysis has emerged as a potential method to accurately and objectively to assess HER2 ITH.AbstractAmplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is associated with an adverse prognosis. The introduction of anti-HER2 targeted therapy has dramatically improved the clinical outcomes of patients with HER2-positive breast cancer. Unfortunately, a significant number of patients eventually relapse and develop distant metastasis. HER2 intratumoral heterogeneity (ITH) has been reported to be associated with poor prognosis in patients with anti-HER2 targeted therapies and was proposed to be a potential mechanism for anti-HER2 resistance. In this review, we described the current definition, common types of HER2 ITH in breast cancer, the challenge in interpretation of HER2 status in cases showing ITH and the clinical applications of anti-HER2 agents in breast cancer showing heterogeneous HER2 expression. Digital image analysis has emerged as an objective and reproducible scoring method and its role in the assessment of HER2 status with ITH remains to be demonstrated.