The gourd family (Cucurbitaceae) is an economically important and diverse group of plants, with approximately 1000 species. Previous analysis of hybridization data and nuclear ITS regions in 70 species of 14 genera of the family Cucurbitaceae identified seven putative holobaramins. The present study expands upon this previous analysis by analyzing the sequence similarity of the chloroplast genome between 30 species from Cucurbitaceae. The clustering analysis showed six putative holobaramins: Gynostemma, Gomphogyne+Hemsleya, Cucurbita, Cucumis, Citrullus, Coccinia grandis+Hodgsonia macrocarpa+Lagenaria siceraria+Momordica charantia+Siraitia grosvernorii+Trichosanthes kirilowii. All six groups had a significant p-value except Gomphogyne+Hemsleya. Of these, Gynostemma and Gomphogyne+Hemsleya are two new putative holobaramins. The present study is the second of its kind, after an analysis of chloroplast genomes in Liliales. This study used both hybridization data and chloroplast genome sequences that supported each other's results. Future chloroplast genome studies could be performed on plant groups that have enough species with mitochondrial genomes.