We developed two ligase-dependent probe amplification assays based on rolling circle amplification (RCA) and the ligase-dependent reaction (LDR) to differentiate species of Exophiala targeting the rDNA internal transcribed spacer region. We focused on Exophiala dermatitidis and E. phaeomuriformis, two opportunistic inhabitants of indoor wet cells, and further detected E. heteromorpha, E. xenobiotica, and E. crusticola; 57 reference isolates representing the five species were tested. Depending on the RCA probes used, the sensitivity was 100%, and the specificity ranged from 3.7% to 88.6% (median: 46.1%). In contrast, the sensitivity and specificity of the LDR probes targeting the same isolates were 88.6-100% (median: 95.8%) and 95.4-100% (median: 97.7%), respectively. We analyzed 198 additional environmental isolates representing the same Exophiala species. Overall, the sensitivity and specificity of LDR ranged from 89.7% to 100% (median: 94.1%) and from 93.9% to 100% (median: 96.9%), respectively. The assessment of performance and validation of LDR probes using SYBR Green quantitative polymerase chain reaction revealed high reproducibility and an acceptable range limit, in line with the guidelines of the European Network of GMO Laboratories. In conclusion, the LDR assay was more reliable and less expensive than RCA for species-level identification of Exophiala isolates.