Cucumber mosaic virus (CMV; genus Cucumovirus, family: Bromoviridae) has the widest host range of any known plant viruses. Seven virus isolates, originated from different ornamental plant species and greenhouses, were biologically purified, mechanically inoculated onto test plants and their serological differences were assayed based on reactivity with 11 CMV-specific monoclonal antibodies. Following total RNA extraction, coat protein (CP) coding region of CMV isolates was amplified. Based on biological, serological and phylogenetic analysis, only one isolate belonged to CMV subgroup II and other six isolates were equally distributed among the two IA and IB subgroups. Aphid transmission assay showed that no significant difference was observed between transmission efficiency of CMV subgroups IA, IB and II members by Aphis gossypii. The genetic variation and evolution of CMV in Iran was studied by sequence analysis of the CP gene and comparison with equivalent sequences of isolates from other continents that exhibited low genetic diversity and close evolutionary relationships among isolates in subpopulations. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that most of the amino acid sites were under negative selection and only one site was under positive selection.