The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of SARS-CoV2 is possessed by the three most common variants of concern – B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the COVID-19 pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription polymerase chain reaction assay using allelic discrimination of the spike gene N501Ymutation to screen for potential variants of concern and differentiate them from wild-type SARS-CoV-2. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or wild-type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real time reverse transcriptase polymerase chain reaction assay. Our assay compared to sequencing, the gold standard for SNP detection and lineage identification, demonstrated clinical sensitivity of 100% for all 57 specimens displaying N501Y mutant, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wildtype and mutant. Clinical specificity was 100% in all 103 specimens typed as wild-type, with A23063 identified as wild-type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole genome sequencing which are time-consuming, labor-intensive and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole genome sequencing.