Abstract: Studies on the phylogenetic relationship among species are the theoretical basis of hybridization breeding.Wild Citrullus species play important roles in Citrullus lanatus breeding.However,research on relationships among C.lanatus and its close relatives are limited.Moreover,there are disagreements on the taxonomy of the genus Citrullus.Comparative genomic in situ hybridization is an effective method to analyze phylogenetic relationships.The mitotic metaphase chromosomes of Citrullus ecirrhosus,Citrullus rehmii, Citrullus colocynthis and Citrullus naudinianus were hybridized with labeled genome DNA from C.lanatus.The phylogenetic relationship was revealed among species and a close relationship between C.naudinianus and Cucumis melo was found by comparative genomic in situ hybridization with labeled genome DNA from C.melo.This research will provide a theoretical basis for hybridization breeding between watermelon and its close relatives.