The complete mitochondrial genome of the brown brocket deer Mazama gouazoubira and a set of polymorphic microsatellite markers were identified by 454-pyrosequencing. De novo genome assembly recovered 98% of the mitochondrial genome with a mean coverage of 9-fold. The mitogenome consisted of 16,356 base pairs that included 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and the control region, as found in other deer. The genetic divergence between the mitogenome described here and a previously published report was ∼0.5%, with the control region and ND5 gene showing the highest intraspecific variation. Seven polymorphic loci were characterized using 15 unrelated individuals; there was moderate genetic variation across most loci (mean of 5.6 alleles/locus, mean expected heterozygosity = 0.70), with only one locus deviating significantly from Hardy-Weinberg equilibrium, probably because of null alleles. Marker independence was confirmed with tests for linkage disequilibrium. The genetic variation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the phylogeography and population genetic patterns in M. gouazoubira, particularly in the context of habitat fragmentation in South America.