Background: he growing occurrence of resistant tuberculosis strains against isoniazid drug is alarming in the global population, making the treatment a challenging task. The molecular mechanism of resistance has been found to be associated with mutations in several genes. However, types and frequency of mutations within those genes vary between geographical regions.Objective: Explore the mutation S315 T (katG gene) in isoniazid-resistant and -sensitive clinical isolates of Mycobacterium tuberculosis from Ecuador by sequencing and PCR-RFLP Methods & Materials: Thirty-nine isolates of Mycobacterium tuberculosis were tested for first line drug susceptibility using proportion method. Genotypic analysis of resistance associated with isoniazid was performed by sequencing and PCR-RFLP Results: We found that 79.48% of the strains studied by PCR-RFLP with SatI presented the S315 T substitution. Of katG gene sequenced strains, 55.56% presented the mutation S315 T, 3.7% presented the Q439P mutation, while 40.73% no-show mutation. PCR-RFLP with SatI has 93.33% sensitivity and 66.67% specificity with a diagnostic accuracy of 87% compared to proportional method test. Existing strong concordance between PCR-RFLP-SatI and proportional method test, with high statistical significance. Conclusion:The PCR-RFLP with SatI could be a quick test, simple with greater reliability that could be used for Isoniazid resistance detection of Mycobacterium tuberculosis in surveillance programs.