Summary This study presents the first insights into the genetic diversity and structure of the A merican donkey metapopulation. The primary objectives were to detect the main structural features underlying variability among A merican donkey populations, identify boundaries between differentiated gene pools, and draw the main colonization pathways since the introduction of donkeys into A merica in the 15th century. A panel of 14 microsatellite markers was applied for genotyping 350 A merican donkeys from 13 countries. The genetic structure of this metapopulation was analysed using descriptive statistics and B ayesian model‐based methods. These populations were then compared to a database containing information on 476 individuals from 11 E uropean breeds to identify the most likely ancestral donor populations. Results showed the presence of two distinct genetic pools, with confluence of the two in C olombia. The southern pool showed a unique genetic signature subsequent to an older founder event, but lacked any significant influence of modern gene flow from E urope. The northern pool, conversely, may have retained more ancestral polymorphisms and/or have experienced modern gene flow from S panish breeds. The A ndalusian and, to a lesser extent, the C atalan breeds have left a more pronounced footprint in some of the A merican donkey populations analysed.