In order to investigate the genetic diversity of melon accessions, of 191 melon accessions including thick-skinned, thin-skinned, and wild types, which were collected from the National Mid-term Genebank for Watermelon and Melon (Zhengzhou, China) and USDA-ARS GRIN database, were analyzed with 43 chromosomally-distributed SSR markers. Furthermore, chilling tolerance was evaluated using the chilling-injury indices of melon seedlings under the constant temperature 4 ℃ as well as the leaf-ultrastructure changes before and after the low-temperature treatment. Totally, 366 alleles were detected by the SSR markers, with an average of 8.512 alleles per marker. Mean of observed and expected heterozygosity were 0.074 and 0.704, respectively, while mean of the polymorphism information index was 0.668. The method of UPGMA clustered all the accessions into four groups (I, II, III,and IV). Group I had only two genetically diverse Indian accessions. Group II included 34 wild accessions and 15 think-skinned accessions, all of which were originated from India. Group III comprised of 51 thick-skinned accessions and one wild accessions, which were collected from a wide geographic distribution. The remained 75 thin-skinned accessions, 7 thick-skinned accessions and 6 wild accessions were clustered into group IV, most of which were from East Asia. Furthermore, the Bayesian algorithm assigned the whole accession panel into three subpopulations (P1, P2 and P3), mostly correlated to the three germplasm types, i.e., thick-skinned, wild, and thin-skinned accessions. The coefficient of differentiation and Nei’ genetic distance among the three germplasm types indicated the highest level of genetic differentiation (FST) between the thick-skinned and thin-skinned accessions, following by FST values between wild accessions and thick-skinned accessions or thin-skinned accessions. The rank of the diversity level among the germplasm types were: wild accessions > thick-skinned accessions > thin-skinned accessions. The chilling-injury indices of the three germplasm types tended to follow normal distribution; the chilling tolerance of thin-skinned accessions was largely superior to that of wild or thick-skinned accessions. Leaf ultrastructure showed a slight change observed with the seedlings of the thin-skinned accession ‘Hamasu5’, indicating a high level of chilling tolerance. In contrast, the thick-skinned accession ‘Fenghuang’ presented a heavy chloroplast destruction and an aggravated damage in cell ultrastructure, indicating a high sensitivity to chilling temperature.