Abstract Background Currently, various diagnostic kits are being developed to aid in the rapid diagnosis or detection of SARS-CoV-2, but viruses continue to evolve through mutations resulting in phenotypic changes such as increased infectivity and pathogenicity, or vaccine evasion. Over time, such alterations in sequence may also occur at the annealing sites of the primers and probes in the diagnostic kit, leading to false-negative results. To prevent this, a continuous mutation monitoring is required. The authors performed SARS-CoV-2 Whole Genome Sequencing (WGS) and then compared and monitored the difference between actual experimental and public data from the National Center for Biotechnology Information (NCBI) datasets for each mutant strain. Methods WGS was performed by using GridION (Oxford Nanopore Technologies, UK) on 1500 nasopharyngeal swab SARS-CoV-2 positive samples requested for testing at SCL from January 2020 to December 2022. Experimental data were aligned through epi2me and analyzed using the Nextclade platform (Fig. 1A). For public data, 119 sequences reported in Korea (Fig. 1B) were retrieved from the NCBI database, reanalyzed using Nextclade platform, and compared. Results In numerous samples tested, mutation, insertion, and deletion were found to occur over time. In particular, in the case of Omicron subvariants, a common Gap (region 1; Gap: 11288–11296, region 2; Gap: 28362–28370) were identified in orf1ab (RdRP), the target gene for the majority of existing diagnostic kits, and in N gene (Fig. 1). Therefore, while designing a diagnostic kit, this region should not be included in the annealing site. Conclusion In order to maintain an effective diagnosis and quarantine system for COVID-19, mutations affecting the annealing site of primers and probes must be identified through a continuous genome monitoring. The results obtained from the continuous genome monitoring can be utilized to upgrade the existing commercially available kits as well as to develop new kits.