Karyotype and ribosomal DNA distribution on four Cucurbitaceae species was analyzed through dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes. Chromosome sizes varied slightly among the species with Cucumis sativus relatively the largest (~2.5 ${mu}m$) and Momordica charantia the smallest (~1 ${mu}m$). In Cucumis sativus L. (2n = 14), the 45S rDNA hybridized on the pericentromeric area of five chromosomes (metacentric a, b, c, g, and submetacentric d) and the 5S rDNA on the region proximal to the centromere of the short arm of chromosome e. In Luffa cylindrica (L.) Roem. (2n = 26), the 45S rDNA hybridized on the distal regions of the short arms of five chromosomes (metacentric a, b, c, f, and submetacentric d) and the 5S rDNA on the region proximal to the centromere of the short arm of chromosome e. In Lagenaria siceraria (Molina) Standl. (2n = 22), the 45S rDNA hybridized on the distal regions of the short arms of two chromosomes (submetacentric b and metacentric e) and the 5S rDNA juxtaposed with the 45S rDNA signal in a region proximal to the centromere on chromosome e. In Momordica charantia L. (2n = 22), the 45S rDNA hybridized on the majority of the short arms of two metacentric chromosomes (d and k) and the 5S rDNA on the proximal region of the short arm of chromosome e. The interphase and metaphase rDNA distribution and FISH karyotype analyses of the four species showed the possible fate of rDNAs through the process that lead to the interspecific variations among the cucurbits. These results will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species.