Background: Non-tuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing Mycobacteria (RGM), and rifampicin for Slow-Growing Mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes includingrrl,rrs, andrpoBin NTM isolates from Kenya.Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced forrrs, and the 68 SGM were sequenced forrpoBgenes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene.Results: NTM harboring mutations associated with resistance to at least one of the antibiotics used in the macrolide-based therapy were 23% (28/122). Of these NTM, 10.4% (12/122) had mutations in therrlgene with 58.3% (7/12) comprising RGM and 41.7% (5/12) being SGM. Mutation at position 2058 (A2058G, A2058C, A2058T) of therrlgene was seen for 83.3% (10/12) of NTM, while 16.6% (2/12) harbored a A2059G mutation. Of the 54 RGM included forrrscharacterization, 11.1% (6/54) exhibited mutations  at position 1408(A1408G), while 14.7% (10/68) of the SGM had mutations in therpoBgene at positions S531W, S531L, S531Y, F506L, E509H withM.gastrihaving multiple mutations at positions D516V, H526D and, S531F.Conclusion: We demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya.