Objective: To evaluate the performance of a recently updated rapid molecular diagnostic test, GenoTypew MTBDRplus version 2, designed to detect drug resistance in both acid-fast bacilli (AFB) smear-negative and-positive specimens. DESIGN: Sputum samples from 1128 patients at risk for multidrug-resistant tuberculosis (MDR-TB) were tested using MTBDRplus v2 and compared with reference standard MGITTM 960TM drug susceptibility testing. The relationship of participant human immunodeficiency virus (HIV) status, diabetic status, previous treatment, and smear gradation to the likelihood of obtaining an interpretable result was assessed using logistic regression. RESULT S: The sensitivity and specificity of MTBDRplus v2 for detecting MDR-TB, when compared to a reference standard, were respectively 96.0% (95%CI 93.5-97.6) and 99.2% (95%CI 97.0-99.9) in AFB smear-positive specimens and 82.8% (95%CI 63.5-93.5) and 98.3% (95%CI 89.9-99.9) in AFB smear-negative specimens. A dose-response relationship was observed between the proportion of interpretable test results and AFB smear bacterial load after adjusting for age, sex, body mass index, HIV status, previous treatment and diabetic status. CONCLUS ION: While MTBDRplus v2 performs well among both AFB smear-positive and-negative specimens, smear gradation appears to influence both the probability of obtaining an interpretable result and test sensitivity, indicating a significant association between bacillary load and test performance.