Background and Research Aims: Historical geological events and climatic changes have played important roles in shaping population differentiation and distribution within species. Amazilia rutila (Trochilidae) is a widespread hummingbird species in the tropical dry forest along the Pacific slope and the Yucatán Peninsula in Mexico. Methods: We used mitochondrial DNA sequence, ecological niche modelling and niche divergence tests to determine the effects of major geographic barriers and environmental variability on genetic and niche divergence of A. rutila continental populations. Results: Our results revealed three genetic groups without haplotype sharing corresponding to the distribution of individuals/populations from the Pacific slope W of the Isthmus of Tehuantepec (PAC), in Oaxaca and Chiapas E of the Isthmus of Tehuantepec (CHIS_OAX) and those from the Yucatán Peninsula and Guatemala (YUC). Values of neutrality tests suggest past demographic expansion without effective population size changes over time, and the time since the demographic expansion ranged between 39.4 and 84.45 ka BP. Each genetic group differed in their position in environmental space, with low-to-very limited overlap in the fundamental climatic niche dimensions of all groups analyzed, particularly between YUC and PAC. Analysis of climate differentiation and ecological niche comparisons showed that the environmental space occupied by these mtDNA groups is similar but not identical. Conclusion: We conclude that the genetic differentiation of A. rutila is consistent with a model of population isolation by geographical barriers and environmental differences. Inferences about the consequences of past demographic expansion and isolation underlying intraspecific evolutionary relationships await further study. Implications for Conservation: Our findings highlight the importance of preserving evolutionary significant units of this widespread hummingbird species. Conservation actions must consider intrinsic requirements of evolutionarily distinct populations and the environmental drivers that shape their distributions, maximizing preservation of intraspecific genetic variability and monitoring changes in genetic diversity.