Tarantula spiders of the genus Brachypelma Simon 1891 are the only complete genus of arachnids protected from international trade under CITES law. To better understand the genetic cohesion of spiders within this genus, we evaluated multiple genetic fragments (totalling about 2200 bp) for their ability to recover population sub-structure among wild-caught Brachypelma vagans (Ausserer 1875) from Belize. We used a novel non-lethal method of tissue sampling, by inducing autospasy of the medial leg. This method allowed us to release wild-caught individuals of this protected species after DNA sampling. We used arachnid specific PCR primers to amplify targeted regions of B. vagans DNA, testing various combinations for consistency. We compared mitochondrial fragments from two populations of B. vagans (∼50 km apart) for variation in mitochondrial 16S lrRNA (plus 5′ ND1), CO1, and the nuclear ITS-2 spacer. Both lrRNA-ND1 and CO1 provided congruent estimates of population subdivision, and indicated that lrRNA-ND1 contained the greatest variation. The nuclear ITS-2 was surprisingly short (193 bp) and relatively invariant across B. vagans. While both mitochondrial fragments appear suitable to elucidate population subdivision and historical processes in B. vagans, we suggest that mitochondrial markers may overestimate population division in B. vagans. We conclude that along with valuable inferences from mitochondrial regions, the characterization of population sub-structure in tarantula spiders will be enhanced by other estimates from alternate nuclear fragments.