The genus Passiflora L. consists of approximately 530 widely distributed species, including Passiflora edulis, which has drawn interest because of its commercial and agronomic value. Passiflora cincinnata is another important species owing to its long flowering period and resistance or tolerance to diseases and pests. In the present study, the meiotic segregation and pollen viability of an interspecific hybrid (P. edulis × P. cincinnata) and its parents were analyzed. The genomic contents were characterized using chromomycin A3 (CMA3)/4′-6′-diamidino-2-phenylindole (DAPI) staining, fluorescent in situ hybridization with 5S/45S ribosomal DNA (rDNA), genomic in situ hybridization (GISH), and inter-simple sequence repeat (ISSR) markers. The results indicated the diploid chromosome number for the parents and interspecific hybrid was 2n = 18. We also observed regular meiosis, one pair of 5S rDNA sites, and two pairs of 45S rDNA sites that co-localized with two pairs of CMA3 +/DAPI− bands. The GISH data revealed three distinct chromosomal groups in the hybrid. The genetic origins of the interspecific hybrid, and its relationship with its parents were also confirmed using ISSR markers.