In recent years, numerous studies have demonstrated the significant potential of non-Saccharomyces yeasts in aroma generation during fermentation. In this study, 134 strains of yeast were isolated from traditional fermented foods. Subsequently, through primary and tertiary screening, 28 strains of aroma-producing non-Saccharomyces yeast were selected for beer brewing. Headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and chemometrics were employed to analyze the volatile flavor substances in beer samples fermented using these strains. Chemometric analysis revealed that distinct species of non-Saccharomyces yeast had a unique influence on beer aroma, with strains from the same genus producing more similar flavor profiles. Accordingly, 2,6-nonadienal, 1-pentanol, phenyl ethanol, isoamyl acetate, ethyl caprate, butyl butyrate, ethyl propionate, furfuryl alcohol, phenethyl acetate, ethyl butyrate, ethyl laurate, acetic acid, and 3-methyl-4 heptanone were identified as the key aroma compounds for distinguishing among different non-Saccharomyces yeast species. This work provides useful insights into the aroma-producing characteristics of different non-Saccharomyces yeasts to reference the targeted improvement of beer aroma.