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The dysfunction of α and β cells in pancreatic islets can lead to diabetes. Many questions remain on the subcellular organization of islet cells during the progression of disease. Existing three-dimensional cellular mapping approaches face challenges such as time-intensive sample sectioning and subjective cellular identification. To address these challenges, we have developed a subcellular feature-based classification approach, which allows us to identify α and β cells and quantify their subcellular structural characteristics using soft X-ray tomography (SXT). We observed significant differences in whole-cell morphological and organelle statistics between the two cell types. Additionally, we characterize subtle biophysical differences between individual insulin and glucagon vesicles by analyzing vesicle size and molecular density distributions, which were not previously possible using other methods. These sub-vesicular parameters enable us to predict cell types systematically using supervised machine learning. We also visualize distinct vesicle and cell subtypes using Uniform Manifold Approximation and Projection (UMAP) embeddings, which provides us with an innovative approach to explore structural heterogeneity in islet cells. This methodology presents an innovative approach for tracking biologically meaningful heterogeneity in cells that can be applied to any cellular system.