AimsThis study aimed to characterize yeasts isolated from the environment of artisanal cachaca production and brewer's spent grain-bearing in mind their further application in bioprocesses. Methods and ResultsCell morphology, growth and fermentative parameters, and karyotyping were employed for the selection and grouping of yeast strains. The results showed that from 134 yeast strains studied, 142% exhibited cells with snowflake morphology, which is not appropriate for bioethanol production. The fermentation in sugarcane syrup was carried out with 71 Saccharomyces cerevisiae, 19 Torulaspora delbrueckii, eight Wickerhamomyces anomalus, six Candida parapsilosis, five Pichia mashurica, three Candida intermedia, two Clavispora lusitaniae and one Candida aaseri. Among the most important ethanol-producing strains, T. delbrueckii LMQA BSG 7 and S. cerevisiae LMQA SNR 65 presented biomass yield, ethanol yield and productivity similar or higher than PE-2 and CAT-1 (bioethanol industrial strains). ConclusionsThis study showed a high potential for industrial application of the strains LMQA SNR 65 (S. cerevisiae) and LMQA BSG 7 (T. delbrueckii). It was found that the use of the chromosomal profile is not adequate to qualify yeasts concerning their technological performance. Significance and Impact of the StudyThis study reported yeasts isolated from uncommon sources that present significant characteristics for potential application in bioprocesses.