Mycobacterium avium complex (MAC) is an emerging pathogen leading to public health concerns in developing and developed countries, particularly among immunocompromised individuals and patients with structural lung diseases. Current clinical guidelines recommend combination antibiotic therapy for treating MAC pulmonary disease (MAC-PD). However, the rising prevalence of antibiotic resistance poses significant challenges, including treatment failure and clinical recurrence. A deeper understanding of the mechanisms underlying MAC antibiotic resistance is essential to improve treatment outcomes. This study investigates the genetic variations associated with aminoglycoside resistance in an antibiotic-induced Mycobacterium intracellulare mutant derived from a clinical strain. Whole-genome analysis identified seven mutations in the aminoglycoside-resistant mutant, including single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). Key genetic alterations included a frameshift variant in a gene encoding a secreted protein antigen, missense mutations in rpsL and rsmG, and synonymous and in-frame deletion variants in srfAB and mtrB, respectively. These findings highlight the complex genetic landscape of aminoglycoside resistance in M. intracellulare. Understanding these resistance determinants provides valuable insights for developing diagnostic tools to detect drug-resistant MAC strains and optimizing therapeutic strategies for managing MAC infections in clinical practice.