The sister species to all extant angiosperms, Amborella trichopoda, occupies an evolutionary position that makes it a crucial reference for all genomic studies in plant biology. Our complete genome sequence underway will help in understanding the evolution of key angiosperm traits and provide a baseline to examine genome organization throughout angiosperms. We have developed a Fluorescence In Situ Hybridization (FISH) system for locating physically mapped BACs directly on Amborella’s chromosomes. Probes for nuclear DNA localization were selected on the basis of weak to moderate Southern hybridization signals using an Amborella nDNA probe against a panel of BACs. Three main types of FISH signals were found: dispersed/GISH-like signals, centromeric signals, and strong single-locus signals marking specific chromosome arms. BACs with characteristics of the latter two categories were used to construct a partial karyotype that to-date identifies 6 out of 13 chromosome pairs. To assess the quality of our genome assembly, FISH was used to localize BACs mapped to different ends of in silico determined scaffolds. Finally, FISH using mtDNA and cpDNA BAC probes was applied to assess the possibility of large inserts of organellar DNA into the Amborella nuclear genome. We have also constructed a chromosome module for the TOLKIN (Tree of Life Knowledge and Information Network) web-based application, which provides informatics support for phylodiversity and biodiversity research projects.