Carica papaya L. is a polygamous species with three sex types: male, female and hermaphrodite. However, the sex identification is only possible after flowering, by inspection of the flowers, which takes time, labor and financial resources. Molecular biology researchers have developed a number of genetic markers in an attempt to distinguish the papaya sex before reaching reproductive maturity. In the present study, we aimed to provide a molecular diagnosis of early sexing for the female and hermaphrodite plants of two commercially important Brazilian varieties of C. papaya (‘Golden’ and ‘Rubi’). For this, it was investigated seven sequence characterized amplified region markers previously described in the literature by the polymerase chain reaction technique, and then, it was developed a probe for fluorescence in situ hybridization (FISH) protocol by using a chosen marker as probe. This molecular marker was selected based on its consistently polymorphic banding pattern, which was also demonstrated by FISH analyses: fluorescent signals in hermaphrodite nuclei isolated from leaves of both cultivars, but no detectable intensity fluorescence signal in female nuclei from both cultivars. Once the probe was constructed, this new and reliable technique, based on FISH in nuclei, could be a valuable tool for sexing the papaya seedlings with commercial application in large scale and open the possibility towards automatized diagnostic assay.