Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest . Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing . We hypothesized that a spike genotyping nucleic acid amplification test (NAAT) could facilitate high-throughput variant surveillance . We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing . We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population . Between December 1 , 2020 and March 1 , 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8% . We detected 1,567 L452R mutations (38.7 %), 34 N501Y mutations (0.84 %), 22 E484K mutations (0.54 %), and 3 (0.07 %) E484K+N501Y mutations . The assay had near-perfect (98-100 %) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others . The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021 . We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening . This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.