The TB-ARC project is a massive international collaboration, bringing together researchers and data from several continents from across the globe. In an effort to identify the full complement of naturally occurring mutations responsible for drug resistance in clinical Mycobacterium tuberculosis, we sequenced over 1,800 geographically and phenotypically diverse isolates that have been quantitatively characterized for their resistance to a broad spectrum of first and second line antibiotics. We identified, and experimentally validated, novel mutations (and mutation combinations) that explain previously unexplained, low frequency resistance to first and second line drugs, including cycloserine. We have also shown that, in some countries where drug resistant tuberculosis (DR-TB) is epidemic, resistance emerged at the very beginning of the antibiotic era and that these early mutations have persisted for decades within M. tuberculosis populations and now account for a large fraction of today's DR-TB.