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Spatially resolved analysis of uranium isotopes in small volumes of actinide-bearing materials is critical for a variety of technical disciplines, including earth and planetary sciences, environmental monitoring, bioremediation, and the nuclear fuel cycle. However, achieving sub-nanometer scale spatial resolution for such isotopic analysis is currently a challenge. By using atom probe tomography, a three dimensional nanoscale characterization technique, we demonstrate unprecidented nanoscale mapping of uranium isotopic enrichment with high sensitivity across various microstructural interfaces within small volumes (100 nm3) of depleted and low enriched uranium alloyed with 10 wt % molybdenum with different nominal enrichments of 0.20 and 19.75% 235U respectively. The approach presented here can be applied to study nanoscale variations of isotopic abundances in the broad class of actinide-bearing materials, providing unique insights into their origin and thermo-mechanical processing routes.