A diagnostic method based on Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed for detection of CMV genome directly in crude extracts of both healthy and infected gladiolus tissue using primers from the conserved sequences of CMV RNA-3. RT-PCR resulted in the amplification of 540 bp long fragment of CMV coat protein gene (CMV-CP), as expected in most of the plant parts of infected gladiolus samples. Positive signals in Southern hybridization of amplified fragments with cloned CMV-CP cDNA probe also confirmed the presence of CMV genome in gladiolus.