Objectives: Globally, Multidrug-Resistant Tuberculosis (MDR-TB) remains a public health concern. Rapid identification of MDR-TB using conventional or novel technologies is crucial for effective treatment. Here we assessed the quantity and quality of DNA extracted from MDR-TB isolates, allowing whole-genome sequencing (WGS). Methods: This was a retrospective study on MDR-TB isolates from five studies conducted in Zimbabwe between 2011 and 2019. The isolates were stored under ambient 37 oC and -80 oC temperatures, respectively. These isolates were resuscitated and confirmed to be Mycobacterium tuberculosis (MTB). DNA was extracted using the N-Cetyl-N-trimethylammonium-bromide standard protocol. The concentration (A260nm) and purity (A260/280) of the extracted DNA before WGS (concentration ≥20ng/ul) were compared among the different storage conditions on Stata v15. Results: A total of 85 samples were successfully recovered from 106 retrieved. The overall recovery rate was 80.2%. We found a significant difference (p=0.005) in the concentration of the DNA samples by storage temperatures, with samples stored at -80 oC having the lowest concentration. Similarly, a significant difference (p=0.018) was found in the purity of the DNA (samples within the optimal range of 1.8 ± 0.2) by storage conditions, with 34/39 (87.2%) stored under ambient temperature, 18/20 (90.0%) stored in a 37 oC incubator and 1/4 (25.0%) stored at -80 oC. Conclusions: The better concentration and purity obtained from samples stored at 37 oC and ambient temperatures provide an impetus that such storage conditions could be used in many resource-limited settings where power supplies are a limitation to long storage conditions. J Microbiol Infect Dis 2023; 12(4):31-37.