The risk of acquiring additional drug resistance in strains of multidrug-resistant tuberculosis (MDR-TB) during failure of empiric standardized retreatment regimens is poorly defined. We sought to estimate this risk by comparing drug susceptibility profiles and RFLP patterns of paired MDR-TB isolates collected from 27 patients before and after retreatment failure. Among 23 patients with paired isolates with concordant RFLP patterns, 19 (83%) had become resistant to at least one additional drug after failed retreatment. In this limited group of MDR-TB patients, acquisition of resistance was common during failure of empiric drug regimens. Further study is needed to confirm these findings.