Several species of mycobacteria cause infections in humans. Species identification of clinical isolates of mycobacteria is very important for the decision of treatment and in choosing the appropriate treatment regimen. We have developed a multiplex PCR method that can identify practically all known species of mycobacteria, by determination of single-nucleotide differences at a total of 13 different polymorphic regions in the genes of rRNA and hsp65, in four PCR mixes. To achieve this goal, single-nucleotide differences in these polymorphic regions were used to divide mycobacterial species into two groups, than four, eight, etc., in an algorithmic manner. It was sufficient to reach single species level by evaluating 13 polymorphic regions. Evaluation of the multiplex PCR patterns by observable real-time electrophoresis (ORTE) simplified species identification. This new method may enable easy, rapid, and cost-effective identification of all species of mycobacteria.