Rifampin resistance in Mycobacterium tuberculosis is mainly due to a small number of mutations in the rpoB gene coding for the beta subunit of RNA polymerase. Heteroresistance, which is defined as a mixture of wildtype and resistant subpopulations in the same culture, can influence the sensitivity of molecular tests for determining drug resistance by leading to false negative or indeterminable results. In our laboratory, we identified one heteroresistant strain during sequencing of the rpoB gene mutations, which are responsible for Rifampin resistance in M. tuberculosis. The sequence analysis of this isolate demonstrated the mixture of different strains, and the exact nature of the mutation could not be determined. In order to solve this problem, the possible mutation site was digested with Tsp509I restriction endonuclease, which made us separate the different strains. Sequencing of the separated mutated product revealed the mutation responsible for Rifampin resistance. From this result, we propose that, when the suggested mutation site creates and/or deletes a restriction endonuclease recognition site in heteroresistant populations, restriction endonuclease analysis can be used to ease the determination of resistance mutations by sequencing.