Seventy-one strains were isolated from the River Agrio-Lake Caviahue acidic aquatic environment in Argentina. Strains were isolated mainly from the most acidic section of the river (pH 1.8-2.7). According to the mini/microsatellite-primed PCR technique and physiological data, these strains are representatives of a single novel species of the genus Cryptococcus. Analysis of the D1/D2 region of the large-subunit rRNA gene showed that the strains belong to the order Filobasidiales of the subphylum Agaricomycotina. The novel isolates formed a phylogenetic group with Cryptococcus ibericus, Cryptococcus aciditolerans and Cryptococcus metallitolerans, the most closely related species. This group, which is phylogenetically related to the Gastricus clade, is considered to be an ecoclade due to its peculiar ecology and physiology. The name Cryptococcus agrionensis sp. nov. is proposed to accommodate these isolates, with strain CRUB 1317(T) (=CBS 10799(T)=JCM 15321(T)) as the type strain.