&NA;The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65‐kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogensMycobacterium tuberculosis, Mycobacterium avium‐intracellulare, andMycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, includingM. kansasiiandMycobacterium leprae, in formalin‐fixed paraffin‐embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin‐embedded tissue sections.