The prospects for persistence of bees living in fragmented landscapes is a topic of considerable interest due to bees’ importance as pollinators of agricultural crops and wild plants, coupled with the ubiquity of native habitat loss and evidence that bees may be declining worldwide. Population persistence in fragmented areas depends on dispersal potential and maintenance of gene flow among fragments of habitat. Here we used population genetic techniques to characterize, for two equally abundant orchid bee species that differ in their physiology and ecology, levels of genetic differentiation among fragments of tropical forest in southeastern Costa Rica in a ~200 km2 landscape. We measured population differentiation with ϕPT (an analogue to the traditional summary statistic Fst), as well as two measures that may more accurately reflect the level of differentiation when highly variable loci are used: G’st and Dest. We also calculated pairwise genetic distances among individuals and conducted Mantel tests to test the correlation of genetic and geographic distance, for each species. We found strong differences in genetic structure between the species. Contrary to our expectations, each measure of genetic structure revealed that the larger-bodied species, Eulaema bombiformis, had higher levels of differentiation than the smaller species, Euglossa championi. Furthermore, for Eulaema bombiformis there was a significant positive correlation of genetic and geographic distance while for Euglossa championi there was no significant positive correlation. Our results demonstrate that bee species can have strikingly different levels of gene flow in fragmented habitats, and that body size may not always act as a useful proxy for dispersal, even in closely related taxa.