Hawthorn (Crataegus spp.), a plant widely distributed in temperate and subtropical regions, is valued for its bioactive compounds and diverse health benefits. Known for its remarkable adaptability to various environmental conditions, hawthorn thrives across different altitudes, but these environmental factors, particularly altitude, significantly influence the accumulation of its bioactive substances. This study investigates the effects of altitude on hawthorn’s nutritional, bioactive, and mineral profiles to provide insights into its cultivation and utilization. Through comprehensive analysis of 20 nutritional indicators from high- and low-altitude samples, including essential nutrients, bioactive compounds, and trace elements, multivariate analyses such as Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) revealed clear altitude-driven clustering. While primary nutritional components like dietary fiber, protein, and soluble solids exhibited stability across different altitudes, low-altitude samples showed higher levels of hypericin, quercetin, and rutin, likely due to favorable light and temperature conditions. Conversely, high-altitude samples were enriched in calcium, reflecting adaptations to cold stress and structural needs, while phosphorus content was reduced under cooler conditions. Potassium, iron, zinc, selenium, and strontium levels remained stable, indicating robust metabolic regulation. These findings confirm the significant role of altitude in shaping hawthorn’s bioactive and mineral profiles, providing essential guidance for altitude-specific cultivation practices and tailored processing strategies. By leveraging these insights, the functional and nutritional properties of hawthorn can be optimized, supporting its sustainable application in the food and health industries.