Nontuberculous mycobacteria (NTM) are ubiquitous bacteria widely distributed in natural environments. Among these species, Mycobacterium intracellulare is an opportunistic pathogen that causes pulmonary NTM infection in a wide range of hosts. Recently, human infection of NTM has been increasing. Furthermore, dog infection is also increased. Our previous study examined NTM distribution in soils, identifying a significant proportion of multidrug-resistant M. intracellulare isolates. To elucidate the diversity of environmental strains, we conducted whole-genome analysis of M. intracellulare isolates. Also, virulence and immune responses of the M. intracellulare isolates were analyzed by comparison of the genomic sequences and infection to mouse alveolar macrophages, AMJ2-C11. Single-molecule sequencing revealed discrepancies between conventional identification methods and complete sequence phylogeny. Although some isolates were identified as M. intracellulare using housekeeping genes, complete sequence phylogeny suggested that they should be reclassified as M. paraintracellulare. Thus, we further analyzed virulence genes of environmental isolates compared to clinical strains, including M. intracellulare-related strains (M. paratintracellulare, M. chimaera, M. yongonese). Some isolates exhibited unique genes related to GPL locus and copper exporter genes. To assess the potential impact on host immune response, we infected mouse alveolar macrophage with 6 environmental isolates and M. intracellulare ATCC13950 as a type strain. Most environmental isolates induced host immune responses similar to clinical strain. Additionally, the number of intracellular bacteria in macrophages is higher than the clinical strain. Apoptotic cell counts increased after environmental isolates infection, while necrotic cell counts remained comparable to the type strain (p<0.05). Moreover, environmental isolates induced higher levels of proinflammatory cytokines (TNF-α, IL-6 and TGF-β) production than those with the type strain. Other 7 cytokines were not detected in the . Whole-genome sequencing of M. intracellulare isolates not only helps to construct databases of complete sequences for M. intracellulare and related strains but also provides important insights into virulence factors and pathogenesis. This study was carried out with the support of the “Cooperative Research Program of Center for Companion Animal Research (Project No. PJ01398501)” RDA, Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea.