The Cryptococcus species complex contains two closely related basidiomycetous yeasts: Cryptococcus neoformans and C. gattii, which cause cryptococcosis in humans and other animals. The species and varieties are characterized, by different clinical, epidemiological, biochemical and molecular features. The currently used identification methods are either time-consuming or not anymore commercially available. However, a rapid, sensitive and robust assay for the detection of these pathogens is vital for early diagnosis and appropriate treatment decisions. To overcome those limitations, four padlock probes targeting species-specific single nucleotide polymorphisms at the internal transcribed spacers (ITSs) of the RNA gene locus were developed and applied during isothermal hyperbranched rolling circle amplification (HRCA). The probes were tested against 99 samples, including 94 clinical cryptococcal cultures, three closely related Cryptococcus species, and two clinical specimens. The use of the padlock probes and the combination of probe signal amplification by HRCA provided a quick and sensitive assay for the accurate identification of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. HRCA was also useful to detect hybrids, when they were heterozygous at the ITS locus. The H RCA results were in agreement with previous genotyping data based on PCR fingerprinting, amplified fragment length polymorphism. and ITS sequencing.