BACKGROUND: Rapid culture and identification of mycobacteria is important to effective treatment and management of patient. We made in-house biphasic media and designed identification system by multiplex PCR/PCR-RFLP. We tried to compare the report time of new system with that of conventional one. METHODS: A total of seven hundred and forty specimens sent for tuberculosis culture in a tertiary care hospital were inoculated into biphasic media which was composed of Ogawa media and Middlebrook 7H9 broth. When growth was detected, Mycobacterium tuberculosis and nontuberculous mycobacteria were identified by cord formation in AFB stain and multiplex PCR using IS6110 and hsp65 gene. Diagnostic algorithm for mycobacteria was established by PCR-RFLP with 13 species, 58 mycobacteria. We tried to identify 14 M. tuberculosis and 18 nontuberculous mycobacteria by that system. RESULTS: Eighty-four mycobacteria (11.4%) out of 740 specimens isolated. The mean report time of new system was 21+/-8.2 day, while that of conventional one was 31+/-8.3 day (P<0.005). Algorithm capable of identification 13 mycobacteria species was made. It showed 19 RFLP patterns and 7 of them were new ones which have not been described. The result of identification for 32 mycobacteria by PCR-RFLP was identical with that of biochemical method. CONCLUSIONS: Culture and identification of mycobacteria by biphasic media and Multiplex PCR/PCR- RFLP was a easy, rapid and cost-effective method in the area with the high prevalence rate of tuberculosis.