Abstract Background The New World leaf-nosed bats (Phyllostomids) exhibit a diverse spectrum of feeding habits and innovations in their nutrient acquisition and foraging mechanisms. However, the genomic signatures associated with their distinct diets are unknown. Results We conducted a genomic comparative analysis to study the evolutionary dynamics related to dietary diversification and specialization. We sequenced, assembled, and annotated the genomes of five Phyllostomid species: one insect feeder (Macrotus waterhousii), one fruit feeder (Artibeus jamaicensis), and three nectar feeders from the Glossophaginae subfamily (Leptonycteris yerbabuenae, Leptonycteris nivalis, and Musonycteris harrisoni), also including the previously sequenced vampire Desmodus rotundus. Our phylogenomic analysis based on 22,388 gene families displayed differences in expansion and contraction events across the Phyllostomid lineages. Independently of diet, genes relevant for feeding strategies and food intake experienced multiple expansions and signatures of positive selection. We also found adaptation signatures associated with specialized diets: the vampire exhibited traits associated with a blood diet (i.e., coagulation mechanisms), whereas the nectarivore clade shares a group of positively selected genes involved in sugar, lipid, and iron metabolism. Interestingly, in fruit-nectar–feeding Phyllostomid and Pteropodids bats, we detected positive selection in two genes: AACS and ALKBH7, which are crucial in sugar and fat metabolism. Moreover, in these two proteins we found parallel amino acid substitutions in conserved positions exclusive to the tribe Glossophagini and to Pteropodids. Conclusions Our findings illuminate the genomic and molecular shifts associated with the evolution of nectarivory and shed light on how nectar-feeding bats can avoid the adverse effects of diets with high glucose content.