Omicron variants of SARS-CoV-2 has been dominantly distributed worldwide since December 2021. Sequence analysis has revealed a 9 base pair (bp) nucleotide deletion located in N gene was found in Omicron strains but not in other prevalent variants. Based on this feature, we have developed a highly specific and sensitive real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for differentiating Omicron lineages from other SARS-CoV-2. With this unique design of primers and probes, we consider this approach could be applied to identify particular genome feature for future SARS-CoV-2 variant characterization.