Wild relative species of domesticated crops are useful genetic resources for improving agronomic traits. Cytogenetic investigations based on chromosome composition provide insight into basic genetic and genomic characteristics of a species that can be exploited in a breeding program. Here, we used FISH analysis to characterize the ploidy level, chromosome constitution, and genomic distribution of 5S and 45S ribosomal DNA (rDNA) in four wild Cucurbitaceae species, namely, Citrullus lanatus All species were diploids, except for T. kirilowii, which included hexa-, octa-, and decaploid cytotypes (2n = 6x = 66, 8x = 88, and 10x = 110). All species have small metaphase chromosomes in the range of 2-5 µm. The 45S rDNA signals were localized distally compared to the 5S rDNA. C. lanatus var. citroides and M. japonica showed one and two loci of 45S and 5S rDNA, respectively, with co-localization of rDNA signals in one M. japonica chromosome. S. angulatus showed two co-localized signals of 5S and 45S rDNA loci. The hexaploid T. kirilowii cytotype showed five signals each for 45S and 5S rDNA, with three being co-localized. This is the first report of hexaploid and decaploid cytotypes in T. kirilowii. These results will be useful in future Cucurbitaceae breeding programs.