The incidence of watermelon chlorotic stunt disease and the molecular characterization of the Jordanian isolate of Watermelon chlorotic stunt virus (WmCSV-[JO]) are described in this study. Symptomatic leaf samples obtained from watermelon (Citrullus lanatus Thunb.), melon (Cucumis melo L.), squash (Cucurbita pepo), cucumber (Cucumis sativus L.), and bottle gourd (Lagenaria siceraria) plants were tested for WmCSV-[JO] infection by PCR. The virus could be detected in 8 melon and 87 watermelon samples obtained from Ghor Assafi (southern part of Jordan Valley). Three samples collected from Mafraq (eastern part of Jordan) were found mixed infected with WmCSV-[JO] and Squash leaf curl virus. The full-length DNA-A and DNA-B genomes of WmCSV-[JO] were amplified, and sequences were deposited in the GenBank under accession numbers EU561237 and EU561236, respectively. Sequence analysis reveals that WmCSV-[JO] is closely related to other virus isolates from Israel (WmCSV-[IL]), Yemen (WmCSV-[YE]), Iran (WmCSV-[IR]), Lebanon (WmCSV-[LB]), and Sudan (WmCSV-[SD]). DNA-A of WmCSV-[JO] showed highest nucleotide identity (99.42%) with WmCSV-[IL], while DNA-B had highest nucleotide identity (95.52%) with WmCSV-[YE]. Data of this study demonstrate that digestion of DNA-B genome of WmCSV isolates with ApaI enzyme can discriminate between these isolates at the molecular level. Infectious clones of WmCSV-[JO] were constructed and agroinoculated to Nicotiana benthamiana plants. Inoculated plants developed mild disease symptoms 4 weeks post inoculation, while watermelon plants biolistically inoculated with WmCSV-[JO] developed characteristic mottling, yellowing and severe leaf curling symptoms 3 weeks post inoculation.