This study investigated the effects of single-strain and mixed-culture fermentation on the flavor profile and quality of highland barley whiskey. A yeast strain (LC3) was isolated from pit mud of traditional Chinese Baijiu, and compared with commercial whiskey yeast (W19) in both single and mixed fermentation systems. Highland barley and malt were mixed at a 1:1 ratio and fermented under controlled conditions. Flavor evaluation was conducted using a combination of electronic nose (E-nose) analysis, sensory panel assessment with flavor wheel development, and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOFMS). The results showed that LC3 produced superior ethanol yield and enhanced ester formation compared to W19. Mixed-culture fermentation (LC3 + W19) significantly increased the concentration and diversity of key volatile compounds, including ethyl hexanoate and ethyl decanoate, contributing to enhanced fruity and floral notes while suppressing undesirable volatiles. E-nose PCA analysis revealed distinct odor profiles among the fermentation groups, supporting the synergistic effect of mixed cultures. This study demonstrates that combining pit mud–derived and commercial yeast strains provides a viable strategy to enhance flavor complexity, ester content, and overall sensory quality of highland barley whiskey. These findings offer a theoretical basis and practical guidance for optimizing whiskey fermentation and improving the quality of highland barley–based distilled spirits.