Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by PPR virus (PPRV). PPR is endemic in Asia, the Middle East and across large areas of Africa and is currently targeted for global eradication by 2030. The virus exists as four different lineages that are usually limited to specific geographical areas. However, recent reports of spread of PPRV, in particular of lineage IV viruses to infection-free countries and previously PPR endemic areas are noteworthy. A rapid and accurate laboratory diagnosis and reports on its epidemiological linkage for virus spread play a major role in the effective control and eradication of the disease. Currently, molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) are usually used for diagnosis of PPR while the sequencing of part of the nucleocapsid gene is usually carried out for the viral lineage identification. However, it is difficult to diagnose and sequence the genetic material if the animal excreted a low level of virus at the initial stage of infection or if the PPRV is degraded during the long-distance transportation of samples to the reference laboratories. This study describes the development of a novel nested RT-PCR assay for the detection of the PPRV nucleic acid by targeting the N-protein gene, compares the performance of the assay with the existing conventional RT-PCR and also provides good-quality DNA suitable for sequencing in order to identify circulating lineages. The assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I–IV) of PPRV. This assay provides a solution with an easy, accurate, rapid and cost-effective PPR diagnostic and partial genome sequencing for use in resource-limited settings.