Chromosome identification is critical for cytogenetic research and will accelerate studies on genetic variation and breeding, especially for those species with relatively little sequence information. So far, no reliable cytological landmarks have been developed to distinguish individual chromosomes in melon. In this study, using FISH (fluorescence in situ hybridization) combined with comparative genome information, we selected 21 cucumber fosmids anchored by SSR markers as chromosome-specific cytological markers for melon chromosomes. Moreover, with the help of melon centromeric satellite DNA repeats CentM, 45S rDNA and 5S rDNA, sequential FISH with 3 sets of multi-fosmid cocktails were conducted on the same metaphase cell, which allowed us to simultaneously identify each of the 12 metaphase chromosomes of melon and a standardized melon karyotype of somatic metaphase chromosomes was constructed. Finally, we compared the distribution of 21 FISH-mapped fosmids between melon and cucumber chromosomes, which allows a better understanding of the evolutionary process shaping these 2 species. Our study provides a basis for cytological characterization of the melon genome and comparative genomics of Cucurbitaceae.