•The genus Passiflora has gaps in delimitations and relationships between species.•The chromosome number of 19 species was revised and defined for further nine.•Chromosome classification was re-evaluated for nine species and established for 19.•The karyogram was assembled for first time for 24 species.•The nuclear DNA content was revisited for 22 species and expanded for 19. The Passiflora genus, which is recognized for its economic and ecological potential, has been studied in botanical, systematic and evolutionary aspects. Currently, Passiflora is represented by five subgenera subdivided into 16 supersections, 31 sections and 13 series. However, their delimitations and relationships are still conflicting. We applied classical cytogenetics to revise the chromosome number of 19 species and to define it for further nine. The classification of chromosome pairs was re-evaluated for nine species and established for further 19. For 24 species, the karyogram was assembled for the first time, and for the other four it was updated. Using flow cytometry, the nuclear DNA content was revisited for 22 species and expanded for 19. Chromosome number 2n = 12 was found for the subgenus Decaloba, as well as 2n = 18 and 2n = 20 for Passiflora, 2n = 24 for Astrophea and Deidamioides, and the unpublished 2n = 48 for P. contracta (subgenus Deidamioides). Metacentric and submetacentric chromosomes predominated in all karyotypes, except in P. lindeniana and P. arborea (subgenus Astrophea), which some acrocentric pairs occur. The nuclear 2C value ranged from 0.59 pg (P. capsularis) to 5.46 pg (P. quadrangularis), with differences exceeding 925 %. The karyotypes and nuclear genome size of the genus Passiflora supported the current subgeneric division. The evaluated characters also corroborate with the hypothesis of x = 6 as the ancestral chromosome number and the diversification by polyploidy and ascendant dysploidy. Furthermore, the cytogenetic and genome size data were complementary to systematic of the genus.