The tropical fruit plant Papaya (Carica papaya L.) can be used as an excellent model species for molecular cytogenetic mapping owing to its relatively small diploid genome of 372 Mbp with nine pairs of chromosomes. Using a set of reported 12 linkage group-specific bacterial artificial chromosomes as FISH probes, we developed a pachytene chromosome-based high resolution karyotype for the hermaphrodite plant genome of papaya cultivar ‘Sunup’, which includes heterochromatin distribution and centromere location. According to the FISH mapping results, the 12 linkage groups were specifically assigned to the nine pairs of meiotic chromosomes. In addition, pachytene chromosome-based cytogenetic analysis demonstrated that the papaya male-specific region of the Y chromosome (MSY) spans the centromere and covers approximately 13% of the Yh chromosome. MSY and its corresponding X chromosome region are cytologically differentiated by the presence of four knob-like heterochromatin structures within MSY. DNA sequences associated with the MSY-specific heterochromatic knobs are highly divergent and heavily methylated compared with the sequences in the corresponding X chromosomal domains, indicating that DNA methylation and heterochromatinization might be responsible for the early stage evolution of the sex chromosome. The high-resolution karyotype and cytological characterization of MSY in papaya will serve as a foundation for future comparative cytological research of papaya and its wild relatives.