BACKGROUND: Nontuberculous mycobacteria (NTM) cause pulmonary and extrapulmonary infections. Although isolation of NTM from clinical specimens has increased nationally, few studies delineated the molecular characteristics of extrapulmonary NTM. METHODS: Extrapulmonary isolates were collected by four Emerging Infections Program sites from October 2019 to March 2020 and underwent laboratory characterization, including matrix-assisted laser desorption ionization-time of flight mass spectrometry, Sanger DNA sequencing, and whole genome sequencing. Bioinformatics analyses were employed to identify species, sequence types (STs), antimicrobial resistance (AR), and virulence genes; isolates were further characterized by phylogenetic analyses. RESULTS: Among 45 isolates, the predominant species were Mycobacterium avium (n=20, 44%), Mycobacterium chelonae (n=7, 16%), and Mycobacterium fortuitum (n=6, 13%). The collection represented 31 STs across 10 species; the most common ST was ST11 (M. avium, n=7). Mycobacterium fortuitum and Mycobacterium abscessus isolates harbored multiple genes conferring resistance to aminoglycosides, beta-lactams, and macrolides. No known AR mutations were detected in rpoB, 16S, or 23S rRNAs. Slow-growing NTM species harbored multiple virulence genes including type-VII secretion components, adhesion factors, and phospholipase C. CONCLUSION: Continued active laboratory- and population-based surveillance will further inform the prevalence of NTM species and STs, monitor emerging clones, and allow AR characterization.