The genus Magnolia includes over 250 species that range in ploidy level from diploid to hexaploid. Although there is basic information on ploidy levels of various species, sampling has been limited and little information on specific cultivars and hybrids is available. The objective of this research was to determine relative genome sizes and relationships to ploidy levels among a diverse collection of species, hybrids, and cultivars using flow cytometry. Nuclei were extracted, stained with 4′, 6-diamidino-2-phenylindole (DAPI), and analyzed using a flow cytometer. Relative genome sizes were determined using Pisum sativum as the reference genome. Genome size was calibrated with ploidy level for species with documented chromosome numbers. Relative genome size for a given ploidy level varied significantly among most taxonomic sections indicating these groups have undergone considerable genomic divergence. These data also indicate it is desirable to calibrate ploidy level with relative genome size for each section separately. Within a section, relative 2C genome sizes, for a given ploidy level, had narrow ranges and could be used to clearly distinguish between euploid levels. Genome size estimates, determined with DAPI or propidium iodide fluorochromes, varied (by 0% to 14%) as a function of species and base pair (bp) composition. Both methods were suitable for determining euploid level. Base pair composition of representative Magnolia species ranged from 61.6% to 63.91% AT. Genome sizes and ploidy levels are presented for a broad range of species and hybrids within genus Magnolia. This information also provides further insight into reproductive biology, substantiation of numerous hybrids and induced polyploids, and comparison of methods for determining genome size that will help facilitate the development of improved hybrids in the future.