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We analyzed spectral cubes of Callisto’s leading and trailing hemispheres, collected with the NIRSpec Integrated Field Unit (G395H) on the James Webb Space Telescope. These spatially resolved data show strong 4.25μm absorption bands resulting from solid-state¹²CO₂, with the strongest spectral features at low latitudes near the center of its trailing hemisphere, consistent with radiolytic production spurred by magnetospheric plasma interacting with native H₂O mixed with carbonaceous compounds. We detected CO₂rovibrational emission lines between 4.2 and 4.3μm over both hemispheres, confirming the global presence of CO₂gas in Callisto’s tenuous atmosphere. These results represent the first detection of CO₂gas over Callisto’s trailing side. The distribution of CO₂gas is offset from the subsolar region on either hemisphere, suggesting that sputtering, radiolysis, and geologic processes help sustain Callisto’s atmosphere. We detected a 4.38μm absorption band that likely results from solid-state¹³CO₂. A prominent 4.57μm absorption band that might result from CN-bearing organics is present and significantly stronger on Callisto’s leading hemisphere, unlike¹²CO₂, suggesting these two spectral features are spatially antiassociated. The distribution of the 4.57μm band is more consistent with a native origin and/or accumulation of dust from Jupiter’s irregular satellites. Other, more subtle absorption features could result from CH-bearing organics, CO, carbonyl sulfide, and Na-bearing minerals. These results highlight the need for preparatory laboratory work and improved surface–atmosphere interaction models to better understand carbon chemistry on the icy Galilean moons before the arrival of NASA’s Europa Clipper and ESA’s JUICE spacecraft.