Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P. edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P. cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA+ bands only in the satellites, but no DAPI+ bands. Fluorescence in situ hybridisation (FISH) showed that in P. cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P. cacaoensis is a more basal species than P. edulis. The cytogenetic data indicate that P. cacaoensis is closely related to P. edulis, but is a different species.