Flowers from members of the genus Leucadendron have colourful bracts and long vase life that make them highly desirable cut-flowers. Breeding programs based on interspecific hybridization would encounter difficulty if pre- or post-fertilization barriers exist in the distant crosses. Embryo rescue is one of the commonly used approaches to overcome post-fertilization barriers in wide hybridization. In this study, intersectional and intersubsectional hybridization of Leucadendron was attempted. Observation of pollen-pistil interactions revealed that post-zygotic rejection was the main reason for the incompatibility of the crosses, therefore embryo rescue was adopted and a protocol was developed to raise the hybrids. To better understand the genome structure in the genus, karyotypes of selected species were analyzed. Chromosome examination indicated that all (27) Leucadendron species examined were diploid and had a chromosome number of 2n = 26. The chromosomes were small in size and had predominantly median to submedian centromeres. The karyotypes of the species were rather symmetrical and seemed to be primitive according to Stebbins' karyotype classification. DNA based PCR-RFLP and RAMP markers were developed to identify Leucadendron hybrids at an early age. RAMP analysis showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP. The occurrence of PCR recombination also proved to be a troublesome issue when using the PCR-RFLP method, whereas the clarity of the interpretion of the RAMP method was not influenced by PCR recombination. Interspecific hybridization in a breeding program can provide valuable information on grouping of the species for systematic purposes. Regression analysis between cross success rate and cpDNA character difference revealed that there was a highly significant correlation between them. Patterns of success for intersectional hybridizations in Leucadendron were generally consistent with current taxonomic hypotheses regarding the sectional division of the genus. Success was generally lower for intersectional crosses than for intrasectional crosses.