Rifampin is a key component of standard short-course first-line therapy against Mycobacterium tuberculosis (MTB). Rifampin monoresistant MTB, previously a rare phenomenon, is now being reported at increasing rates worldwide. We report a mutation in the rpoB region leading to low level rifampin monoresistance in a cluster of HIV-positive patients. All rifampin monoresistant isolates identified from 2004 to 2006 underwent susceptibility confirmation, sequencing of rpoB and genotyping. Three patients were found to have a previously undocumented 3 base pair insertion at codon 525 in the rpoB region. The earliest initial case was infected with fully susceptible MTB. Disease relapse occurred 7 months later with a genotypically identical MTB isolate, showing acquired rifampin monoresistance. MTB isolates from 2 subsequent patients showed primary rifampin monoresistance with an identical genotype to the index case. Patients with rifampin monoresistant MTB tend to have poorer outcomes than those with fully susceptible strains. Risk factors for the development of rifampin monoresistance include co-morbid HIV infection and previously treated tuberculosis. HIV infection has been associated with malabsorption of anti-tuberculous medications leading to sub-therapeutic levels of administered drugs. These factors may have played a role in the development of this previously undocumented mutation.