Background: COVID-19 vaccination-related lymphadenopathy is a frequent imaging finding that may be indistinguishable from malignant nodes and can lead to diagnostic difficulties in patients with cancer or healthy individuals on cancer screening. However, no prospective trials regarding COVID-19 vaccination-related lymphadenopathy following a booster shot have been conducted. The purpose of this study was to determine the incidence and imaging characteristics of COVID-19 vaccination-related axillary lymphadenopathy and assess the recovery period following a booster shot. Methods: We prospectively enrolled healthy women working at St. Luke’s International Hospital, who would receive the third shot of the Pfizer-BioNTech COVID-19 vaccine between December 6 and 28, 2021. Women with a history of cancer, atopic dermatitis, auto-immune disease, or axillary surgery were excluded. All participants underwent ultrasound (US) examinations for the bilateral axilla at baseline (prior to the third shot), early phase (1–3 days after the shot), and late phase (6 weeks after the shot) if lymphadenopathy was detected at the early phase. We evaluated the incidence and US characteristics of lymphadenopathy. As for US characteristics mimicking a malignant node, focal cortical thickening, absence of the echogenic hilus, and vascularity were examined. In this study, abnormal lymphadenopathy was defined as [1] an increase in the short-axis size by more than 2 mm compared with the baseline, [2] an increase in the number of nodes with short-axis diameter more than 5 mm, and [3] demonstrating US characteristics mimicking malignant nodes. Results: A total of 100 women were enrolled in this study. The median age was 41 years (range 23–63). Abnormal axillary lymphadenopathy on the vaccinated side was observed in 59% of participants in the early phase and 8% in the late phase. In the contralateral axilla, abnormal lymphadenopathy was observed in 1% of participants in the early phase and 2% in the late phase. The median short-axis size of ipsilateral abnormal lymphadenopathy was 7.6 mm in the early phase and 5.7 mm in the late phase. In the early phase, US characteristics mimicking malignant nodes were observed, including focal cortical thickening in 54% of participants, absence of the echogenic hilus in 16%, and hypervascularity in 33%. Conclusions: COVID-19 vaccination-related axillary lymphadenopathy indistinguishable from malignant nodes was observed in more than half of the participants compared with the baseline, which improved in most cases within 6 weeks after the latest booster shot. To avoid a diagnostic conundrum, patients with breast cancer should be vaccinated on the arm contralateral to the cancer side. It is recommended that non-urgent imaging screening for the axilla should be scheduled after 6 weeks following the latest vaccination.