We analyzed the phylogenetic relationships of all eleven currently recognized species of large Artibeus using the cytochrome b mitochondrial gene. The topology from a maximum parsimony analysis included: (1) A. obscurus and A. planirostris as sister species with successively basal lineages of (2) A. amplus, (3) a clade with A. lituratus and A. intermedius, (4) A. jamaicensis, (5) a clade of A. inopinatus sister to A. hirsutus and A. fraterculus, (6) A. fimbriatus, and (7) the most basal lineage of A. concolor. The individual species were monophyletic and well supported by bootstrap and decay values. The monophyletic clade of ((((obscurus planirostris) amplus) (lituratus intermedius)) jamaicensis) was also highly supported, although some of the interspecific relationships were less so. Contrary to previous hypotheses of species limits based on a presumed intergradation in body size, A. jamaicensis and A. planirostris do not form a monophyletic group, refuting their conspecificity and supporting an earlier study concluding that these two taxa represent separate morphological populations. An analysis with A. jamaicensis and A. planirostris constrained as sister-taxa resulted in a tree 8 steps longer. In addition, the low genetic pair-wise difference between A. lituratus and A. intermedius (2.8% with Kimura-2 parameters) warrants closer examination of their species limits.