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Magnolias are characteristic tree species of the Tropical Montane Cloud Forest (TMCF) in Mexico, an ecosystem that is highly threatened by habitat fragmentation and climate change. In this study, based on DNA sequences from five regions (chloroplast: trnT-trnL, trnK5-matK, trnS-trnG, rpl32-trnL, nuclear: ITS) and seven nuclear microsatellite markers, we aimed to delineate species boundaries between two-endemic species of the TMCF, Magnolia pedrazae and Magnolia schiedeana, and to estimate levels of genetic structure and diversity among populations. Phylogenetic and haplotype network analyses for the chloroplast and ITS regions did not support genetic differentiation as two distinctive species. Results from Bayesian and multivariate cluster analyses based on microsatellite loci showed high genetic differentiation across most populations, which was consistent with a strong and significant pattern of isolation by geographical distance. We found moderate to high levels of population genetic diversity, but it was lower in small populations relative to large populations. Our results suggest a contemporary decrease of genetic connectivity among populations, likely as a consequence of the current decline of suitable TMCF habitat. Managing landscape connectivity among remnant Magnolia populations within protected natural parks and surroundings, and with emphasis of small populations, would be key for the species conservation.