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Rapid and accurate diagnosis of infections is fundamental to containment of disease. Several monkeypox virus (MPV) real‐time diagnostic assays have been recommended by the CDC; however, the specificity of the primers and probes in these assays for the ongoing MPV outbreak has not been investigated. We analyzed the primer and probe sequences present in the CDC recommended MPV generic real‐time PCR assay by aligning those sequences against 1730 MPV complete genomes reported in 2022 worldwide. Sequence mismatches were found in 99.08% and 97.46% of genomes for the MPV generic forward and reverse primers, respectively. Mismatch‐corrected primers were synthetized and compared to the generic assay for MPV detection. Results showed that the two primer‐template mismatches resulted in a ~11‐fold underestimation of initial template DNA in the reaction and 4‐fold increase in the 95% LOD. We further evaluated the specificity of seven other real‐time PCR assays used for MPV and orthopoxvirus (OPV) detection and identified two assays with the highest matching score (>99.6%) to the global MPV genome database in 2022. Genetic variations in the primer‐probe regions across MPV genomes could indicate the temporal and spatial emergence pattern of monkeypox disease. Our results show that the current MPV real‐time generic assay may not be optimal to accurately detect MPV, and the mismatch‐corrected assay with full complementarity between primers and current MPV genomes could provide a more sensitive and accurate detection of MPV. [ABSTRACT FROM AUTHOR]