Background and Aims: Certification programs of plant propagating materials rely on faster, cheaper and more importantly sensitive and reliable methods for detection of systemic pathogens as indicated in national and/or international health standards of plant propagating materials. Reverse transcription-polymerase chain reaction (RT-PCR) has been documented as an alternative assay for certification of plant propagating materials. RT-PCR has been shown to be necessary for olive certification due to the inefficiency of other methods in detecting viruses. The object of the present study was the optimization of a multiplex RT-PCR assay for simultaneous detection of Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV) and Cucumber mosaic virus (CMV) together with the plant mRNA as internal control for olive certification programs in Iran.Materials and Methods: Total RNA was extracted from olive tissues infected by ArMV, CLRV and CMV as well as from healthy plants and subjected to cDNA synthesis by M-MuLV reverse transcriptase. Simplex, duplex and multiplex RT-PCR (s-, d-, and mRT-PCR) were optimized for amplification of target genes. Amplified fragments were further sequenced for evaluation of the accuracy of the assays.Results: Genomic fragments of ArMV, CLRV, CMV and the plant internal control (Nad 5 gene) were successfully amplified in all assays. The sequence information as well as application of the developed method on samples derived from different origins revealed the accuracy of all assays in olive certification schemes.Conclusion: Results from the developed s-, d-, and mRT-PCR assays revealed RT-PCR as an excellent assay for olive certification. Moreover, coamplification of Nad5 gene fragment suggested that was a robust marker for analyzing the accuracy of the developed RT-PCR as shown previously in other crops as well.