Fluoroquinolones (FQs) are important second-line bactericides used in the treatment against Mycobacterium tuberculosis (Mtb) by inhibiting DNA gyrase. Chromosomal mutations within the Quinolone Resistance Determining Regions (QRDRs) of the gyrase subunits, referred to as canonical mutations, are the primary cause of FQ resistance. However, 10-30% of the observed resistance is seen in clinical isolates without such canonical mutations, suggesting that other mechanisms of resistance are present. This study analyzed the gyrase subunits gyrA and gyrB, as well as 64 efflux pump genes and their predicted promoters, in 347 clinical Mtb isolates from India, Moldova, Philippines and South Africa. Twenty-nine isolates had no known resistance conferring mutations although they were phenotypically resistant; no susceptible isolates had canonical mutations. Five novel variants within the gyrA QRDR and 2 outside the gyrB QRDR were discovered in resistant isolates. The structural effect of certain gyrA variants was investigated in-silico by comparing the molecular dynamics simulations of the protein-drug complexes of wild-type and mutant proteins. These simulations indicate that the gyrA G112A mutation could play a possible role in resistance. The alternative efflux pump mechanism was studied by analyzing the variant patterns in efflux pump genes and their promoters across resistant and susceptible isolates. Variants that were present exclusively in FQ resistant clinical isolates in these regions were identified. These associations could provide insights into how variants in efflux pumps and their promoters can turn on/off these pumps, thus affecting resistance. These results warrant further investigation into key gyrase mutations and efllux pump gene/promoter variants as independent mechanisms of resistance.