Repetitive DNA sequences with variability in copy number or/and sequence polymorphism can be employed as useful molecular markers to study phylogenetics and identify species/chromosomes when combined with fluorescence in situ hybridization （FISH）. Cucumis sativus has three variants, Cucumis sativus L. var. sativus, Cucumis sativus L. var. hardwickii and Cucumis sativus L. var. xishuangbannesis. The phylogenetics among these three variants has not been well explored using cytological landmarks. Here, we concentrate on the organization and distribution of highly repetitive DNA sequences in cucumbers, with emphasis on the differences between cultivar and wild cucumber. The diversity of chromosomal karyotypes in cucumber and its relatives was detected in our study. Thereby, sequential FISH with three sets of multi-probe cocktails （combined repetitive DNA with chromosome-specific fosmid clones as probes） were conducted on the same metaphase cell, which helped us to simultaneously identify each of the 7 metaphase c