Background: Drug resistance continues to threaten recent global progress toward tuberculosis (TB) control. Rapid and accurate detection of resistance is imperative for successful treatment outcomes and reduction in transmission of resistant strains. Methods: Global frequency and distribution of mutations associated with isoniazid resistance were characterized through an exhaustive systematic literature review. The diagnostic performance of a newly redesigned line probe assay (LPA) among both Acid Fast Bacilli (AFB) smear positive and negative specimens was evaluated using cross-sectional data from a large multisite diagnostic study. Additionally, the association of clinical characteristics and valid test results (susceptible or resistant) was assessed using logistic regression. Individual probe results from two LPAs were compared to both sequencing data and phenotypic susceptibility results, and the relative contribution of absent wild type probe hybridization to the detection of phenotypic resistance was determined by comparing the areas under the receiver operating characteristic curves. Results: Globally 64% of isoniazid resistant specimens harbor the katG315 mutation and 19% harbor the inhA-15 mutation, together these two mutations are associated with 80% of isoniazid resistance. Sensitivity and specificity of the LPA for multidrug-resistant TB detection were 95.1% (95%CI 92.6, 96.9) and 99.1% (95%CI 97.1, 100.0) respectively, among both AFB smear positive and negative specimens. Sensitivity for isoniazid resistance was significantly lower among AFB smear negative specimens at 81.6% (95%CI 65.1, 91.7) compared to 94.9% (95%CI 92.4, 96.6) among AFB smear positive specimens. Additionally, AFB smear gradation was significantly associated with valid test results after controlling for HIV status, diabetic status, body mass index, and history of previous treatment. The inclusion of absent wild type probe hybridization in resistance classification for LPAs significantly improved detection of both rifampicin and flouroquinolone phenotypic resistance (P<0.001). Conclusions: Variation in mutation frequency justifies local monitoring of resistance associated mutations. The newly redesigned LPA performed well among both AFB smear positive and negative specimens, however, bacillary load influenced both the probability of obtaining an interpretable result and test sensitivity. The evaluation of individual LPA probe results demonstrated that current LPA resistance classification algorithms successfully differentiate between phenotypically susceptible and resistant specimens.