Cucurbits are very diverse group of plant species that are cultivated throughout the world under various conditions and with different purposes. Economically important crops include cucumbers, melons, watermelons and squash. Various pathogens affect these plants, in which viral diseases are more devastating and of particular importance due to their difficult control methods. In this study, HC-Pro protein was evaluated to better understand the evolutionary relationship of Watermelon mosaic virus (WMV) isolates. Contaminated plant samples were collected from several watermelon farms in Golestan province in the summer of 2017 and were subjected to serological assay by specific WMV antibodies. In order to sequence the HC-Pro protein, two pairs of primers were designed with the help of the Primer-BLAST, available on the NCBI server, to amplify two overlapping fragments (each 850 nucleotides). After editing and assembling the raw sequences, the complete sequence of HC-Pro was obtained with length of 1360 nucleotides (454 amino acids). The results of BLAST indicated that our isolate with 98% identity is most closely related to the French and Italian isolates. For phylogenetic studies, all complete sequences of watermelon mosaic virus (85 isolates) available in GenBank were aligned with our isolate by MAFFT software. Among the available sequences, only a one complete sequence of Iranian isolate was identified. Initially, sequences were analyzed by RPD4 to remove evolutionary noise. A total of 8 isolates were identified and eliminated as recombinant, among which there was also Iranian isolate. Therefore, our sequenced isolate is the only representative of Iranian isolates. The evolutionary model of GTR + G + I was obtained using SMS software. Phylogenetic analysis was performed based on this model using PhyML3. Also, the phylogenic tree was rooted by the Soybean mosaic virus. Based on the results, the isolates of Watermelon mosaic virus are divided into four groups. Similar to the results of BLAST, the Iranian isolate was located in a group with European isolates. French and Korean isolates are scattered across all groups, and a Chinese isolate (KX926428) is separated from others at the base of the tree. These evidences confirm the history of watermelon culture in the world, a native of Africa, introduced to Asian countries, especially China, then to Europe and American. Also, the genetic diversity of Korean isolates is directly linked to the import of watermelons from western countries to South Korea. Therefore, the evidence suggests the European ancestors for our Iranian isolate. This is the first study of the HC-Pro protein of Watermelon mosaic virus in Iran.