Passionflower (Passiflora incarnata L.) is used in phytotherapy as a mild sedative and anxiolytic agent. In the literature it is clear this plant shows considerable qualitative and quantitative variability with respect to its content of C-glycosyl flavones, some of which are used as marker compounds for extracts. Analysis of plant material cultivated in Australia revealed two chemically distinct groups; hence an investigation was carried out to determine whether distinct intraspecific chemotypes exist in this species. Eleven P. incarnata samples were analysed by HPLC, LC-MS and two different TLC methods. The samples fell into two distinct groups with respect to their C-glycosyl flavone profile, with little within-group variation. One chemotype was dominated by isovitexin and schaftoside/isoschaftoside, as is most widely reported in the literature for this species. The other chemotype was characterized by a high level of swertisin, with low levels of schaftoside/isoschaftoside. The two chemotypes are readily identified by both HPLC and TLC. Although the compounds responsible for the therapeutic activity of P. incarnata are yet to be identified, phytomedicines should be made with the accepted isovitexin chemotype until the pharmacological implications of chemotypical differences are understood.