Morus nigra L. (Moraceae) is one of Iran’s notable native species, valued for its medicinal and nutritional properties, particularly in its foliage. The species is widely distributed across Iran, from arid lowlands to high-altitude regions, where environmental conditions can influence plant metabolite composition. In this study, leaves from 15 Iranian M. nigra varieties were analyzed to investigate their flavonoid profiles and the effect of altitude on metabolite accumulation. Ultrahigh-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF/MS) was employed for precise qualitative and quantitative analysis. Seven flavonoids were identified, including isoquercitrin, kaempferol methyl ether glucuronide, quercetin, and rutin, confirming the reliability of the analytical method. Quantitative analysis revealed that flavonoid accumulation increased with altitude, with samples from the highest elevation exhibiting the greatest total flavonol content (1026.1 ± 79.4 mg/g). These findings highlight that geographical factors, particularly altitude, play a significant role in determining flavonoid content in M. nigra leaves. The study provides new insights into the distribution of bioactive flavonoids in mulberry leaves and underscores their potential as stable, industrially valuable ingredients with antioxidant and functional properties. The results also offer guidance for optimizing cultivation and harvesting strategies to maximize flavonoid yield, supporting the development of mulberry leaf extracts for food, pharmaceutical, and cosmetic applications.
