The phylogenetic relationships and chromosomal evolution of the diverse tropical genus Passiflora (Passifloraceae) are explored using data from two chloroplast markers: the rpoC1 intron and the trnL/trnT spacer region. A survey of the presence or absence of the rpoC1 intron in 136 species representing 17 of Killip's (1938) 22 subgenera of Passiflora and four other genera in the Passifloraceae revealed intron losses in 46 taxa. A minimum of two losses were confirmed by a parametric bootstrap approach on sequence data from the trnL/trnT chloroplast non-coding region for 61 taxa. The results of phylogenetic analyses of the trnL/trnT sequence data support the reduction of Killip's 22 subgenera to four as proposed in a new classification system by Feuillet and MacDougal (2004). The monophyly of the ‘n=6’ and ‘n=9’ chromosomal and morphological groups is strongly supported. In addition, these data indicate that Passiflora biflora, or closely related species, is the likely continental sister to the red-flowered Caribbean taxa, while P. auriculata is weakly supported as the New World sister to the Old World Passifloras. Finally, character optimization of chromosome numbers on the phylogenetic tree supports x=12 as the base chromosome number for Passiflora.