Three isolates of Cucumber mosaic virus (CMV) from lily plants showing mosaic and distortion symptoms were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) using primers specific to Cucumovirus genus namely, LK-CMV, LK4-CMV, and LK5-CMV. Restriction enzymes patterns of the RTPCR products revealed that the lily isolates belonged to subgroup IA of CMV. In terms of biological properties, the lily isolates have highly similar but distinct pathogenicity as reported in other lily strains and ordinary strains of CMV. To characterize the molecular properties, cDNAs containing coat protein (CP) gene and 3`` non-coding region (NCR) of RNA3 for the isolates were cloned and their nucleotide sequences were determined. The CP similarity (218 amino acids) was highly homologous (>97%) with that of subgroup I CMV strains. However, an additional 20-nulcleotide long segment was only present in 3`` NCR of lily isolates, which form an additional stem-loop RNA structure. By using chimeric construct exchange cDNA containing 3``NCR of LK-CMV into the full-length cDNA clone of RNA3 of Fny-CMV, this additional segment may prove to be significant in the identification and fitness of the virus in lily plants. The pathology of zucchini squash infected by F1F2L3-CMV, a pseudorecombinant virus was showed to change drastically the severe mosaic and stunting symptom into a mild chlorotic spot on systemic leave, compared with Fny-CMV. To delimit the sequence of RNA3 affected the pathology, various RNA3 chimeras were constructed between two strains of CMV. The symptom determinants of F1F2L3-CMV were mapped to the positions amino acid 234, 239, and 250 in 3a movement protein (MP). RNA3 chimeras changed the sequences encoding three amino acids were resulted in alteration of systemic symptom.