Although guanacos (Lama guanicoe) are widely distributed throughout much of their historic range, they are often restricted to small, isolated populations that are at risk of inbreeding and loss of genetic diversity. Here we document and interpret baseline patterns of genetic variation in a guanaco population inhabiting in the Bolivian Chaco that is threatened by hunting and habitat degradation. The region, once open grasslands, is dominated by dense shrubs, the result of livestock grazing. The most-recent census identified only 74 individuals and is designated as at 'risk of extinction' by the Bolivian government. We assessed the population's genetic health and uniqueness from the genetic patterns of 16 microsatellite loci, the Hyper-variable Domain I of the mitochondrial Control Region and SRY gene with 29 fecal samples. We identified 19 male and 10 female unique individuals. Microsatellite variation (Ho = 0.64) was similar to larger, less-isolated populations. However, the estimated effective population size was low and consistent with the continuing loss of allelic variation. The two observed mtDNA haplotypes are common in other L. g. guanicoe populations. Although we documented moderate genetic diversity, gene flow among Bolivian and Paraguayan guanaco Chaco populations should be assessed and integrated into explicit conservation management plans.