Background: Preliminary evidence suggests that human epidermal growth factor receptor 2 (HER2) expression may play a role in tumor cell proliferation in prostate cancer. Minner et al (Clin Cancer Res, 2010) identified a prevalence of ~ 20% HER2 expression in prostate cancer. The aim of the study is to assess the prevalence and discordance rate of HER2 expression in prostate cancer patients with multiple prostate biopsies using immunohistochemistry (IHC) at a Veterans Affairs hospital. Methods: A single-center, retrospective chart review of at least 30 patients with prostate cancer that had two prostate biopsies based on data collected from the Computerized Patient Record System (CPRS) and Veterans Information Systems and Technology Architecture (VISTA). Demographic and laboratory data including age, race, Gleason score, and stage of cancer were collected under IRB-approved waivers for consent and HIPAA authorization. Formalin-fixed paraffin embedded (FFPE) prostate tumors were evaluated for IHC analysis by the Washington DC Pathology Department. HER2 expression was assessed using the VENTANA anti-HER2/neu (4B5) rabbit monoclonal primary antibody and scored as 0, 1+, 2+, and 3+. HER2 scores of 0 and 1+ to 3+ were categorized as HER2-negative and HER2-positive, respectively. The primary endpoints are the prevalence and discordance rate of HER2 expression in paired prostate samples. Results: In our predominately African American (94%) cohort of 32 patients with a mean age of 60 years, overall HER2 prevalence was 25% (n = 8) and 56% (n = 18) in the primary and secondary biopsies, respectively. The mean time between biopsies was 41 weeks and overall HER2 discordance was 56% (n = 18). Discordance from HER2-negative to HER2-positive was 38% (n = 12), while discordance from HER2-positive to HER2-negative was 6% (n = 2). The remaining discordance were from HER2-low to HER2-positive or vice-versa. The most common variance between primary and secondary biopsies was observed as +1 HER2 scoring category (n = 11). No significant associations were found between HER2 discordance and Gleason score or stage of cancer . Conclusions: In our cohort of patients with prostate cancer, HER2 expression was observed in 25% of initial biopsies and over 50% of secondary biopsies. Discordance of HER2 expression was found in over 50% of patients. A few patients had initial HER2-positive biopsies and subsequent HER2-negative biopsies. The majority of HER2 discordance involved patients who had initial HER2-negative biopsies and secondary HER2-positive biopsies with most showing +1 difference in IHC scores. This study could lead to the consideration of utilizing multiple prostate cancer biopsies to determine the eligibility for anti-HER2 therapy in future trials.