Aim To explore whether environmental factors are correlated with genetic and morphometric differences in the widely distributed bat species Tadarida brasiliensis. Location North America and Central America. Methods We used an extensive sampling comprising 131 localities that represent heterogeneous environments across the Nearctic and Neotropical regions. Museum specimens were examined and 25 craniodental characters were recorded. Individuals were genotyped at one mitochondrial locus (mtDNA) and nine nuclear loci (nDNA). Clustering and phylogenetic analyses were used to identify differentiated groups. Environmental variables and PCA-env approaches were used to determine the climatic niche and to measure the niche overlap, equivalence and similarity between groups. Mantel tests between genetic groupings and environmental variables, dispersal costs, Euclidean geographical distances and niche overlap were performed. Results We identified six genetic groups within Central and North American T. brasiliensis based on nDNA. The most strongly differentiated group, in both nDNA and mtDNA, was located in central Mexico. Morphometric data showed that individuals from populations in Florida are slightly larger than the others. Niche overlap was detected among Neotropical groups but not among Nearctic groups. The currently recognized subspecies were not recovered as distinct groups with either genetic or morphometric data. Main conclusions Our approaches suggest that environmental niche variation may help shape the distribution of genetic variation across heterogeneous landscapes, particularly in widely distributed species. Environmental niche analyses suggest that genetic differences between migratory and non-migratory groups of T. brasiliensis may be promoted by climatic variation throughout the Nearctic and Neotropical regions. In addition, genetic and morphometric analyses do not support the current subspecies classification of T. brasiliensis in North and Central America, which should be abandoned.