M.Sc. / Africa's cycads (66 species and 2 subspecies in two endemic genera: Encephalartos and Stangeria) are extremely endangered with four species extinct in the wild and 80% threatened (CR, EN, or VU) with all included in CITES Appendix 1. Although South Africa has some of the world’s strictest cycad legislation, these plants are still under threat from illegal collection for horticulture and medicine especially where they are seized in an unidentifiable condition. Currently developed legislation demands accurate identification for permit issue. Ex situ conservation of genetic and locality based diversity is paramount. Furthermore, taxonomically many species of unknown origin are difficult to identify especially when diagnostic characters are absent. Species delimitation and numbers are uncertain with field observations often contradicting current understanding. DNA barcoding can assist with all the above-mentioned scenarios. In the current study all proposed DNA barcoding regions (matK, rbcLa, psbA-trnH, and nrITS) along with several additional regions were tested on ~350 samples from which a phylogeny of 63 of the 65 Encephalartos species was also constructed. Results show general good amplification and sequencing success of proposed barcoding regions, although a shift to specialist primers was made in several cases. Genetic variation however was extremely low as is resolution at species level, even when multi-locus barcodes were employed. Results obtained from the phylogenetic analyses show an increase in resolution at both species and higher levels compared to previous work and as such several new groupings are delimitated. Each species grouping is characterised by shared, derived morphological, ecological, and geographic characters and when compared to previous phylogenetic studies are supported to some extent. The current study provides the first step towards a much-needed monograph and revision of the entire genus Encephalartos.