Background To develop and evaluate rapid, molecular-based drug susceptibility tests (DST) for extensively drug resistant tuberculosis (XDR-TB), we assembled a phenotypically and genotypically diverse collection of M. tuberculosis (Mtb) isolates from patients evaluated for drug resistance in four high-burden countries. Methods Mtb isolates from India (n=111), Moldova (n=90), the Philippines (n=96), and South Africa (n=103) were selected from existing regional and national repositories to maximize phenotypic diversity for resistance to isoniazid, rifampin, moxifloxacin, ofloxacin, amikacin, kanamycin and capreomycin. MGIT-960 was performed on viable isolates in one laboratory using standardized procedures and drug concentrations. Genetic diversity within drug-resistance phenotypes was assessed. Results Nineteen distinct phenotypes were observed among 400 isolates with complete DST results. Diversity was greatest in the Philippines (14 phenotypes) and least in South Africa (9 phenotypes). Nearly all phenotypes included multiple genotypes. All sites provided isolates resistant to injectable but susceptible to fluoroquinolone drugs. Many patients were taking antibiotics to which their current infection was resistant. Discussion Diverse phenotypes for XDR-TB-defining drugs, including resistance to fluoroquinolone and/or injectable drugs in rifampin-sensitive isolates indicate that rifampin-sensitivity does not ensure effectiveness of a standard four-drug regimen. Thus, rapid, low-cost DST assays for first- and second-line drugs are needed.