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This study aims to genomically elucidate six isolates of rapidly growing non-tuberculous mycobacteria (RGM) derived from Siamese fighting fish (<i>Betta splendens</i>). These isolates had previously undergone phenotypic and biochemical characterization, antibiotic susceptibility testing, and in vivo virulence assessment. Initial DNA barcoding using the 16S rRNA sequence assigned these six isolates to five different species, namely <i>Mycobacterium chelonae</i> (BN1983), <i>M. cosmeticum</i> (BN1984 and N041), <i>M. farcinogenes</i> (SNSK5), <i>M. mucogenicum</i> (BN1956), and <i>M. senegalense</i> (BN1985). However, the identification relied solely on the highest percent identity of the 16S rRNA gene, raising concerns about the taxonomic ambiguity of these species. Comprehensive whole genome sequencing (WGS) and extended genomic comparisons using multilocus sequence typing (MLST), average nucleotide identity (ANI), and digital DNA–DNA hybridization (dDDH) led to the reclassification of BN1985 and SNSK5 as <i>M. conceptionense</i> while confirming BN1983 as <i>M. chelonae</i> and BN1984 and N041 as <i>M. cosmeticum</i>. Notably, the analysis of the BN1956 isolate revealed a potential new species that is proposed here as <i>M. mucogenicum</i> subsp. <i>phocaicum</i> sp. nov. Common genes encoding “mycobacterial” virulence proteins, such as PE and PPE family proteins, MCE, and YrbE proteins, were detected in all six isolates. Two species, namely <i>M. chelonae</i> and <i>M. cosmeticum</i>, appear to have horizontally acquired T6SS-II (<i>clpB</i>), catalase (<i>katA</i>), GroEL (<i>groel</i>), and capsule (<i>rmlb</i>) from distantly related environmental bacteria such as <i>Klebsiella</i> sp., <i>Neisseria</i> sp., <i>Clostridium</i> sp., and <i>Streptococcus</i> sp. This study provides the first draft genome sequence of RGM isolates currently circulating in <i>B. splendens</i> and underscores the necessity of WGS for the identification and classification of mycobacterial species.