Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that in turn facilitate species identification and breeding programs. Tandem repeats (TRs) like the 45S rDNA, 5S rDNA and telomeric repeats are ubiquitous in nuclear genomes and are good cytogenetic markers for karyotyping. In this study, we analyzed the karyotypes of three exotic cucurbit species, namely Cucumis melo var. flexuosus (L.) Naudin (2n = 24), Melothria pendula L. (2n = 24) and Trichosanthes anguina L. (2n = 22), based on the cytogenetic distribution of the 45S, 5S and Arabidopsis-type telomeric TRs through triple-color fluorescence in situ hybridization. T. anguina had larger chromosomes (3.2-5.4 µm) compared to C. melo var. flexuosus and M. pendula (1.5-2.2 µm and 1.8-2.5 µm). One and two pairs of 5S and 45S rDNA signals were observed in C. melo var. flexuosus, respectively; while M. pendula and T. anguina had four and three pairs of 45S rDNA, respectively, and two pairs of 5S rDNA. Co-localized signals of 5S and 45S rDNA were observed in M. pendula and T. anguina, but not in C. melo var. flexuosus. Telomeric repeats were observed at chromosome ends of all chromosomes. This information will be useful in future cytogenomic and phylogenetic studies facilitating cucurbit breeding programs.