Genome sequencing with next-generation sequence (NGS) technologies can now be applied to organisms pivotal to addressing fundamental biological questions, but with genomes previously considered intractable or too expensive to undertake. However, for species with large and complex genomes, extensive genetic and physical map resources have, until now, been required to direct the sequencing effort and sequence assembly. As these resources are unavailable for most species, assembling high-quality genome sequences from NGS data remains challenging. We describe a strategy that uses NGS, fluorescence in situ hybridization, and whole-genome mapping to assemble a high-quality genome sequence for Amborella trichopoda, a nonmodel species crucial to understanding flowering plant evolution. These methods are applicable to many other organisms with limited genomic resources.