The connection between genetic mutations linked to delamanid resistance and phenotypic resistance remains unclear. We assessed the phenotypic effects of delamanid-resistant mutations in the Mycobacterium tuberculosis H37Rv strain through gene disruption using homologous recombination and complementation tests. Delamanid resistance was assessed by determining the minimum inhibitory concentration (MIC) via the 7H9 microdilution method. Sanger sequencing identified mutations, and conservation of the mutated residues was predicted through multiple sequence alignments of orthologs. A total of 116 isolates with MIC ≥0.025 μg/mL were analyzed, among which mutations were identified in the ddn and fbiA genes. Isogenic strains were generated based on these mutations. The ddn or fbiA isogenic strains with Ala77Val, Gly81Ser, Asn25fs, and Leu104Phe in fbiA had MICs ≥0.8 μg/mL, indicating resistance. In contrast, the ddn isogenic strain with Pro12Ala had an MIC of 0.012 μg/mL, showing susceptibility, while Gly96Asp in fbiA had an MIC of 0.1 μg/mL, indicating resistance. All mutations, except for Pro12Ala, were conserved in the protein sequences of both FbiA and Ddn and their mycobacterial orthologs. The characterization of these mutations provides insights into the mechanisms of delamanid resistance, which may inform the development of optimized treatment strategies.