We designed a multiplex qPCR to differentiate monkeypox virus clades. In evaluations using clinical samples collected in the UK and Nigeria, the assay had an overall sensitivity of 78% (95% CI: 67.67% to 86.14%) and specificity of 94% (95% CI: 80.84% to 99.30%); for samples under Ct35 sensitivity was 98% (95% CI: 91.72% to 99.96%) and specificity was 94% (95% CI: 80.84% to 99.30%).