Coffee (Coffea arabica), produced and marketed in Ecuador and worldwide, can be organoleptically improved by means of microorganisms such as well-characterized yeasts. This study aimed to isolate and characterize yeasts from three postharvest fermentation processes (i.e., Natural aerobic at room temperature; Carbonic maceration with a CO₂ atmosphere at room temperature; and Carbonic refrigerated maceration with a CO₂ atmosphere to 10 °C) of coffee fruits in Ecuador. Phenotypic and molecular analyses were conducted on 329 yeast isolates obtained from coffee farms in Loja, Olmedo, and Gonzanamá. Three universal media were used for yeast isolation diversity, and phenotypic characterization included morphology, sugar fermentation, salt tolerance, and ethanol resistance. Molecular characterization involved DNA analysis. The isolated diversity was classified into 12 morphotypes, nine distinct biochemical groups and nine genetic species. Only six species (i.e., Kurtzmaniella quercitrusa, Hanseniaspora opuntiae, Pichia. kluyveri, Torulaspora delbrueckii, T. quercuum, and Wickerhamomyces anomalus) identified phylogenetically corresponded to the designated morphotypes. But surprisingly, nine genetic species matched with the nine biochemical groups determined phenotypically analyzed using principal component analysis (PCA). Most of this diversity was found in the coffee plantation located in Gonzanamá, in contrast to Olmedo and Loja, without statistical significance (p value: 0.08295). On the other hand, the richness is not similar statistically (p value: 0.02991) between postharvest fermentation treatments. The findings suggest that the application of biochemical tests is useful for species determination, although morphological data may be ambiguous. Notably, Pichia kluyveri, detected in this study, holds potential for biotechnological evaluation in coffee fermentation processes.