In the Iberian Pyrite Belt (IPB), acid rock drainage gives rise to aquatic habitats with low pH and high concentrations of heavy metals, a situation that causes important environmental problems. We investigated the occurrence and diversity of yeasts in two localities of the IPB: São Domingos (Portugal) and Rio Tinto (Spain). Yeast isolation was performed on conventional culture media (MYP), acidified (pH 3) media (MYP3), and on media prepared with water from the study sites (MYPw). The main goal of the study was to determine the structure of the yeast community; a combination of molecular methods was used for accurate species identifications. Our results showed that the largest fraction of the yeast community was recovered on MYPw rather than on MYP and MYP3. Twenty-seven yeast species were detected, 48% of which might represent undescribed taxa. Among these, an undescribed species of the genus Cryptococcus required low pH for growth, a property that has not been observed before in yeasts. The communities of S. Domingos and R. Tinto showed a considerable resemblance, and eight yeast species were simultaneously found in both localities. Taking into consideration the physicochemical parameters studied, we propose a hierarchic organization of the yeast community in terms of high-, intermediate-, or low-stress conditions of the environment. According to this ranking, the acidophile yeast Cryptococcus sp. 5 is considered the most tolerant species, followed by Cryptococcus sp. 3 and Lecytophora sp. Species occurring in situations of intermediate environmental stress were Candida fluviatilis, Rhodosporidium toruloides, Williopsis californica, and three unidentified yeasts belonging to Rhodotorula and Cryptococcus.