Two regions of rpoB associated with rifampin resistance were sequenced in 29 rifampin-resistant (determined by the proportion method) isolates of Mycobacterium tuberculosis obtained from patients from three counties in Hungary. Of the 29 resistant strains, 27 had a mutation in either the 81-bp region (26 strains) or the N-terminal region (1 strain), while the other 2 strains had no mutations in either region. The locations and frequencies of the mutations differed from those previously reported. The most common mutation in this study, D516V, was found in 38% of the Hungarian strains, a frequency 2 to 10 times higher than that found in studies from other countries. These same 29 isolates were also evaluated with the Inno-LiPA Rif. TB test (LiPA), a reverse hybridization assay for the rapid detection of rifampin resistance. Although LiPA detected the presence of an rpoB mutation in 26 of the resistant isolates, the type of mutation could not be determined in 4 isolates because the mutations present were not among those included on the LiPA strip. In addition, a silent mutation in one of the rifampin-susceptible control strains was interpreted as rifampin resistant by LiPA. These findings demonstrate the importance of validating this rapid molecular test by comparison with DNA sequence results in each geographic location before incorporating the test into routine diagnostic work.