1. Ploidy level in plants may influence ecological functioning, demography and response to climate change. However, measuring ploidy level typically requires intensive cell or molecular methods. 2. We map ploidy level variation in quaking aspen, a dominant North American tree species that can be diploid or triploid and that grows in spatially extensive clones.We identify the predictors and spatial scale of ploidy level variation using a combination of genetic and ground-based and airborne remote sensing methods.3. We show that ground-based leaf spectra and airborne canopy spectra can both classify aspen by ploidy level with a precision-recall harmonic mean of 0.75-0.95 and Cohen's kappa of c. 0.6-0.9. Ground-based bark spectra cannot classify ploidy level better than chance. We also found that diploids are more common on higher elevation and steeper sites in a network of forest plots in Colorado, and that ploidy level distribution varies at subkilometer spatial scales. 4. Synthesis. Our proof-of-concept study shows that remote sensing of ploidy level could become feasible in this tree species. Mapping ploidy level across landscapes could provide insights into the genetic basis of species' responses to climate change. K E Y W O R D S adaptation, ploidy level, polyploidy, quaking aspen, reflectance, remote sensing, spectrometry, UAS S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Blonder B, Graae BJ, Greer B, et al. Remote sensing of ploidy level in quaking aspen (Populus tremuloides Michx.). J Ecol. 2020;108:175-188. https ://doi.