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With up to 137 species worldwide, Myotis ,— a genus of small, insectivorous bats are an example for understanding biogeographic and evolutionary processes such as intercontinental colonization, diversification, speciation, and convergent adaptive evolution. Species limits and associated distributions, however, remain poorly delineated across significant portions of this diverse group and encompassing a vast geographic range. Here, we explore how diversity is partitioned across this radiation with a focus on Neotropical species of Myotis , using a barcoding approach (706 bp of the mitochondrial DNA gene cytochrome-b) that includes 544 individual sequences (116 newly generated sequences and 428 sequences from GenBank, including outgroups). Single-locus molecular barcoding is useful for identifying cryptic diversity and establishing hypotheses that can guide investigations of species boundaries and phylogenetic relationships, an especially important initial step in assessing poorly characterized, diverse clades. The global phylogeny produced from these data is consistent with the hypothesized monophyly of the New World clade and places the evolutionary relationships of Neotropical species of Myotis within the context of the Palearctic and Nearctic clades. We then explore species boundaries for 25 of the 36 nominal species of Neotropical Myotis , using 3 alternative species delimitation approaches (1 distance-based ABGD [Automatic Barcode Gap Discovery]; and 2 coalescent-based mPTP [multi-rate Poisson Tree Processes], GMYC [Generalized Mixed Yule Coalescent] model). The mPTP (n = 29 species) and ABGD (n = 26) approaches inferred different numbers of species than that recognized by current Neotropical Myotis taxonomy, whereas GMYC (n = 25) inferred the same number of species. In addition to the original 36 Neotropical nominal species currently recognized, we suggest that taxonomic revisions are needed to assess whether M. oxyotus gardneri and M. nigricans osculatii should be elevated to the species level and whether M. dinellii is distinct from M. levis. With the 7 potentially cryptic species identified herein that require further study, the number of Neotropical Myotis may approach 45 species. Taken together, our results suggest that rather than a classic example of a temperate radiation, there may be more species of Myotis in the Neotropics than Nearctic, Afrotropic, Indomalayan, and Oceanian Realms, with only the Palearctic region showing higher species richness. [ABSTRACT FROM AUTHOR]