Background and Objective: Epidemiological studies indicate that not only the incidence of fungal infections is dramatically on the rise, especially in the immunocompromised hosts, but also the sensitivity of etiological agents to antifungal drugs shows a remarkable reduction. Therefore, early detection at the species level is critically important for proper clinical management. Because standard diagnostic procedures are time consuming, expensive, and less sensitive, PCR-based molecular techniques have been developed. In the present study, we aim to describe a rapid and sensitive technique based on the rolling circle PCR amplification for accurate and fast identification of Cladophialophora carrionii vs. C. yegresii. Materials and Methods: Specific padlock probes were designed based on a single nucleotide polymorphism (SNP) difference in the internal transcribed spacer (ITS) rDNA region of Cladophialophora strains to differentiate between C. carrionii and C. yegresii. The probe sequences are complementary to the target DNA leading to the linker position that after hybridization with the target DNA will be joined together by DNA ligase, form a closed molecule and hybridize to the target DNA for replication at single-temperature conditions. Results: We successfully amplified the target fungi DNA at the species level without any false and negative cross reactivity. The RCA product was visualized on 1.5% agarose gel to clarify the specificity of the probe–DNA template binding. Conclusion: These results demonstrate that RCA is a powerful and accurate tool for discrimination and identification of pathogenic fungi.