Fifty-six Chieh-qua and 7 other cucurbits germplasm resources were collected from South China, subsequently rDNA-ITS amplication and sequencing were performed. With regard to them and ITS sequences of several cucurbitaceous plant downloaded from GenBank, the lengths, variable sites, G C% contents, genetic divergences, homologies, phylogenetics and system position were analyzed by bioinformatic softwares. ITS complete sequences of Chieh-qua and its affinis cucurbitaceae germplasm resources were obtained. The full-length of chieh-qua ITS was 608bp, G C% content were 59.33%( ITS1: 221 bp, G C% content: 60.18%, ITS2: 231bp, G C% content: 63.64%, 5.8S: 157bp, G C% content: 57.96%). The sequence alignment results showed that there were 94 variable sites（ITS1: 47, ITS2: 40, 5.8S: 7）among chieh-qua germplasm resources, some of them had significant species-specific characteristics and could be used as DNA fingerprint markers of Chieh-qua germplasm resources. 56 chieh-qua germplasm materials were clustered into 3 groups based on ITS sequences variance, 12-2-3 was ranked into the first branch, “Heimaojie H2251” was classified into the second branch, and the rest chieh-qua materials and wax gourd were grouped into the third group containing 4 subgroups. Compared with the other cultivated species, the variation of chieh-qua material 12-2-3 was the biggest, the genetic divergence ranged from 4.5 to 5.6. The system position of Chieh-qua was between Indomelothria blumei (GU799496) and Dactyliandra welwitschii (HQ201973), and had the closest relationship with Indomelothria blumei. Based on Mrbayes software analysis, Zehneria thwaitesii(AM981145), Ctenolepis cerasiformis (AM981142), Cucumis melo (AM36377), Dactyliandra welwitschii (HQ20 1973), Trochomeria macrocarpa (AM981141) were the most primordial species, the system evolution order was Cucumis melo(AM36377) → Cucurbita moschata, Trichosanthes yunnanensis, Momordica charantia, Luffa aegyptiaca, Citrullus lanatus (FJ915098), Lagenaria siceraria → Benincasa hispida, Benincasa hispida var. chieh-qua How → Cucumis sativus. The phylogeny of Chieh-qua and its affinis cucurbitaceae germplasm resources was investigated by using ITS sequences and it would provide useful information for their breeding, germplasm DNA fingerprint identification, classification and evolution and comparative analysis, etc.