We investigated the phylogeography and subspecies classification of the ostrich (Struthio camelus) by assessing patterns of variation in mitochondrial DNA control region (mtDNA-CR) sequence and across fourteen nuclear microsatellite loci. The current consensus taxonomy of S. camelus names five subspecies based on morphology, geographic range, mtDNA restriction fragment length polymorphism and mtDNA-CR sequence analysis: S. c. camelus, S. c. syriacus, S. c. molybdephanes, S. c. massaicus and S. c. australis. We expanded a previous mtDNA dataset from 18 individual mtDNA-CR sequences to 123 sequences, including sequences from all five subspecies. Importantly, these additional sequences included 43 novel sequences of the red-necked ostrich, S. c. camelus, obtained from birds from Niger. Phylogeographic reconstruction of these sequences matches previous results, with three well-supported clades containing S. c. camelus/syriacus, S. c. molybdophanes, and S. c. massaicus/australis, respectively. The 14 microsatellite loci assessed for 119 individuals of four subspecies (all but S. c. syriacus) showed considerable variation, with an average of 13.4 (±2.0) alleles per locus and a mean observed heterozygosity of 55.7 (±5.3)%. These data revealed high levels of variation within most subspecies, and a structure analysis revealed strong separation between each of the four subspecies. The level of divergence across both marker types suggests the consideration of separate species status for S. c. molybdophanes, and perhaps also for S. c. camelus/syriacus. Both the mtDNA-CR and microsatellite analyzes also suggest that there has been no recent hybridization between the subspecies. These findings are of importance for management of the highly endangered red-necked subspecies (S. c. camelus) and may warrant its placement onto the IUCN red list of threatened animals.