Background Line probe assay (LPA) is one of the most accurate diagnostic tools for detection of different Mycobacterium species. There are several commercial kits based on line probe assay for detection of Mycobacterium species. Because of their high cost, especially for underdeveloped and developing countries, and the discrepancy of NTM prevalence across geographic regions, it would be reasonable to consider the development of an in-house LPA. The aim of this study was to develop a LPA to detect and differentiate mycobacterial species, and also to evaluate the usefulness of PCR-LPA for direct application on clinical samples. Method One pair of biotinylated primers and fifteen designed DNA oligonucleotide probes were used based on multiple aligned ITS sequences. Specific binding of the PCR amplified products to the probes immobilized on a strip of nitrocellulose membrane was evaluated by hybridization method. Experiments were performed three times on separate days to evaluate the repeatability of the assay. Further, evaluation of the PCR-LPA was carried out directly on 9 clinical samples and cultivated isolates. Results All the fifteen probes used in this study were hybridized specifically to ITS sequences of the corresponding standard species. Results were reproducible for all of the strains in different days. Mycobacterium species of nine clinical specimens and their relevant cultivated isolates were correctly identified by PCR-LPA and confirmed by sequencing. Conclusions In this study, we described a PCR-LPA which is readily applicable in clinical laboratory. This assay is a fast, cost-effective and highly specific method which requires no radioactive materials.