First results for a new atmospheric-pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging source operating at 213 nm laser wavelength are presented. The activation of analytes in the 213 nm MALDI process at atmospheric pressure was evaluated and compared to results for 337 nm MALDI and electrospray ionization using thermometer molecules. Different sample preparation techniques for nicotinic acid, the matrix with the highest ionization efficiency at 213 nm of all tested matrices, were evaluated and optimized to obtain small crystal sizes, homogenous matrix layer sample coverage, and high ion signal gains. Mass spectrometry imaging experiments of phospholipids in mouse tissue sections in positive- and negative-ion mode with different lateral resolutions and the corresponding pre-/post-mass spectrometry imaging workflows are presented. The use of custom-made objective lenses resulted in sample ablation spot diameters of on average 2.9 μm, allowing mass spectrometry imaging experiments to be performed with 3 μm pixel size without oversampling. The ion source was coupled to an orbital trapping mass spectrometer offering high mass resolution (>100.000), high mass accuracy (≤ ±2 ppm), and high sensitivity (single pixel on-tissue tandem MS from 6.6 μm2 ablation area). The newly developed 213 nm atmospheric-pressure MALDI source combines the high mass resolution and high mass accuracy performance characteristics of orbital trapping mass spectrometers with high lateral resolution (pixel size ∼3 μm) mass spectrometry imaging.