We identified 23 cases of
Mycobacterium immunogenum
respiratory acquisition linked to a colonized plumbing system at a new hospital addition. We conducted a genomic and epidemiologic investigation to assess for clonal acquisition of
M. immunogenum
from hospital water sources and improve understanding of genetic distances between
M. immunogenum
isolates. We performed whole-genome sequencing on 28
M
.
immunogenum
isolates obtained from August 2013 to July 2021 from patients and water sources on four intensive care and intermediate units at an academic hospital. Study hospital isolates were recovered from 23 patients who experienced
de novo
respiratory isolation of
M. immunogenum
and from biofilms obtained from five tap water outlets. We also analyzed 10
M
.
immunogenum
genomes from previously sequenced clinical (
n
= 7) and environmental (
n
= 3) external control isolates. The 38-isolate cohort clustered into three clades with pairwise single-nucleotide polymorphism (SNP) distances ranging from 0 to 106,697 SNPs. We identified two clusters of study hospital isolates in Clade 1 and one cluster in Clade 2 for which clinical and environmental isolates differed by fewer than 10 SNPs and had less than 0.5% accessory genome variation. A less restrictive combined threshold of 40 SNPs and 5% accessory genes reliably captured additional isolates that met clinical criteria for hospital acquisition, but 12 (4%) of 310 epidemiologically unrelated isolate pairs also met this threshold. Core and accessory genome analyses confirmed respiratory acquisition of multiple clones of
M. immunogenum
from hospital water sources to patients. When combined with epidemiologic investigation, genomic thresholds accurately distinguished hospital acquisition.