Peste des petits ruminants (PPR) is a globally significant disease of small ruminants caused by the peste des petits ruminants virus (PPRV) that is considered for eradication by 2030 by the United Nations Food and Agriculture Organisation (FAO). Critical to the eradication of PPR are accurate diagnostic assays. RT-qPCR assays targeting the nucleocapsid gene of PPRV have been successfully used for the diagnosis of PPR. We describe the development of an RT-qPCR assay targeting an alternative region (the fusion (F) gene) based on the most up-to-date PPRV sequence data. In silico analysis of the F-gene RT-qPCR assay performed using PCRv software indicated 98% sensitivity and 100% specificity against all PPRV sequences published in Genbank. The assay indicated the greatest in silico sensitivity in comparison to other previously published and recommended PPRV RT-qPCR assays. We evaluated the assay using strains representative of all 4 lineages in addition to samples obtained from naturally and experimentally-infected animals. The F-gene RT-qPCR assay showed 100% diagnostic specificity and demonstrated a limit of detection of 10 PPRV genome copies per μl. This RT-qPCR assay can be used in isolation or in conjunction with other assays for confirmation of PPR and should support the global efforts for eradication.