The genus Aristolochia sensu lato contains over 400 species from warm temperate to tropical regions worldwide. Taxonomic treatments of Aristolochia have been ambiguous and controversial. In a recent cladistic analysis based on morphological characters, it was proposed that the genus should be divided into four genera in two subtribes. To reconsider the systematics of Aristolochia sensu lato, we reconstructed its phylogeny based on nucleotide sequences of the chloroplast rbcL gene and the nuclear-encoded phytochrome A (phyA) gene for 19 representative species and the chloroplast matK gene of over 80 species. All phylogenetic trees produced with the three genes indicate that Aristolochia sensu lato is a monophyletic group, consisting of two lineages that correspond to the subtribes Aristolochiinae and Isotrematinae. The matK phylogeny shows that each of the lineages includes two sublineages. The Aristolochiinae clade is composed of the Aristolochia sensu stricto and Pararistolochia clades, and the Isotrematinae clade of the Isotrema and Endodeca clades. Chromosome numbers, including newly reported counts for 30 species, are predominantly congruent with the phylogeny: the Aristolochiinae clade shows chromosome numbers of 2n = 6, 12, 14, or 16, while the Isotrematinae clade is characterized by 2n = 32. In the Isotrematinae clade, the paralogous relationships of the phyA gene suggest that polyploidization might have occurred.