In recent years molecular data have been used increasingly to estimate phylogenies and aid in species delimitation. We generated and analyzed sequence data for spiny pocket mice (Heteromys) for the mitochondrial gene cytochrome b (1,140 base pairs [bp]) and 2 nuclear gene segments, MYH6 (252 bp) and EN2 (189 bp). We used maximum-parsimony, maximum-likelihood, and Bayesian optimality criteria to estimate relationships among species and provide a framework for using a species-delimitation method to investigate the possibility of multiple species within the widespread H. desmarestianus group. We recovered several well-supported clades within this complex, including H. goldmani, H. oresterus, and H. nubicolens. Incorporating karyotype, allozyme, and morphological data from earlier studies, we found sufficient supporting evidence to justify maintaining H. goldmani, H. oresterus, and H. nubicolens as species and identifying 4 additional clades as candidate species. We present a revised taxonomic arrangement within the genus. The subgenus Xylomys should be retained and be composed of H. nelsoni. The subgenus Heteromys should be divided into 3 species groups: the H. anomalus group (H. anomalus and H. australis) together with H. catopterius, H. oasicus, and H. teleus (insertae sedis); the H. gaumeri group (H. gaumeri); and H. desmarestianus group (H. desmarestianus, H. goldmani, H. oresterus, H. nubicolens, and the 4 candidate species). [ABSTRACT FROM AUTHOR]