Cucumis melo (melon) is a species from the Iberian Peninsula and is included in the family Cucurbitaceae. Despite the knowledge about the physical structure of melon chromosomes, little is known about the intraspecific karyotype diversity of the species. To study karyotype diversity in eight melon accessions, the following methods were used: Giemsa 3% staining; application of CMA3/DAPI fluorochromes; and location of 45S and 5S rDNA sequences by fluorescence in situ hybridization. Conventional staining analysis revealed stability in the chromosome number, with accessions presenting 2n = 24. There were significant variations in the mean chromosome size between accessions, ranging from 0.98 μm to 1.46 μm for accessions A26 and A18, respectively. Scott-Knott clustering distributed the accessions into two groups. GC-rich heterochromatic blocks (CMA3 + /DAPI-) were observed in pericentromeric regions of all chromosomes in the complement. CMA3 + /DAPIblocks were also located in terminal regions, being specific to satellite regions. Hybridization sites of 45S rDNA probes revealed the presence of a chromosome pair with this locus. In addition, 5S rDNA sites revealed a labeled chromosome pair. No quantitative variation was observed in rDNA sites between the accessions analyzed, indicating these markers as ideal for the verification of karyotypic stability in C. melo.