Background: Vaccination is an essential strategy to prevent infection in the SARS-CoV-2 pandemic. However, there are concerns about vaccine efficacy and the impact of vaccination on cancer treatment. Additionally, the emergence of novel variants may affect vaccination efficacy. This multi-center, prospective, observational study investigated the efficacy and impact of vaccination against SARS-CoV-2 variants on treatment among breast cancer patients in Japan. Methods: Breast cancer patients scheduled to be vaccinated with the SARS-CoV-2 vaccine from May to November 2021 were included. They were stratified into five groups according to their cancer treatment: no treatment, endocrine therapy, CDK4/6 inhibitor, chemotherapy, anti-HER2 therapy. Serum samples were collected before the first vaccination and after the second vaccination. Immunoglobulin (Ig)G levels against the SARS-CoV-2 S protein and neutralizing antibody titers against wild-type (WT), alpha (α), delta (δ), kappa (κ), and omicron (ο) variants were measured by ELISA assay. The effect of vaccination on cancer treatment was also investigated. Results: There were 85 eligible patients (no treatment, n = 5; endocrine therapy, n = 30; CDK4/6 inhibitor, n = 14; chemotherapy, n = 21; and anti-HER2 therapy, n = 15) with a median age of 65 years. The overall seroconversion rate of anti-SARS-CoV-2 IgG was 95.3%. The seroconversion rate of the chemotherapy group was 81.8%. The anti-SARS-CoV-2 IgG antibody concentration was positively correlated with the lymphocyte count before vaccination (r = 0.232, p = 0.039). Overall neutralizing antibody titers against each variant were significantly lower than for WT. Overall positive rates of neutralizing antibodies against WT, α, δ, κ, and ο variants were 90.2%, 81.7%, 96.3%, 84.1%, and 8.5%, respectively. A downward trend of neutralizing antibody titers against each variant was seen in chemotherapy and CDK4/6 inhibitor groups compared with other groups. Significant decreases were detected in neutralizing antibody titers against WT, α, and κ variants in the chemotherapy group, and WT and α variants in the CDK4/6 inhibitor group compared with the no treatment group. Withdrawal or postponement of systemic therapy because of vaccination was only observed in one patient. Conclusions: Our data support SARS-CoV-2 vaccination for cancer patients being treated with systemic therapy. However, neutralizing antibody titers against the ο variant were very low even after two vaccinations among patients with or without cancer treatment. Further, a decrease in neutralizing antibody titer was suggested during chemotherapy and CDK4/6 inhibitor, raising concerns about the impact on long-term infection prevention. For these patients, infection-preventive behaviors should be recommended even after vaccination. They will also be good candidates for booster vaccinations. Clinical trial information: UMIN000045527.