Purpose: Heterogeneous human epidermal growth factor receptor 2 (HER2) overexpression in gastric cancer may lead to a misdiagnosis of HER2 status. Accurate assessment of HER2 status is essential for optimal treatment as novel HER2-directed agents are being investigated in various clinical settings. We evaluated the usefulness of HER2 re-assessment following progression on first-line treatment in initially HER2-negative advanced gastric cancer (AGC) patients. Materials and Methods: We enrolled 177 patients with baseline HER2-negative AGC and performed HER2 re-assessment after progression on first-line treatment from February 2012 to June 2016 at Asan Medical Center, Seoul, Korea. The re-assessed HER2 status was analyzed with baseline HER2 status and clinical characteristics. Results: The median age was 54 years (range, 24 to 80 years), and 123 patients (69.5%) were men. Seven patients (4.0%) were HER2-positive on the re-assessment. Patients with baseline HER2 negativity confirmed by a single test (n=100) had a higher HER2-positive re-assessment rate compared to those who had repeated baseline testing (n=77) (5.0% vs. 2.6%). Among the patients with single baseline HER2 testing, the rate was higher in patients with baseline HER2 immunohistochemistry (IHC) 1+ compared to those with IHC 0 (13.4% vs. 3.6%). Conclusion Overall, 4.0% of patients with baseline HER2-negative AGC were HER2-positive on re-assessment, and the HER2-positive re-assessment rate was higher among patients who had a single test at baseline. HER2 re assessment may be considered for initially HER2-negative patients to determine their eligibility for HER2-directed therapy, particularly if their HER2 negativity was determined by a single test, especially if they had a single baseline HER2 IHC 1+ test.