| Summary: | To analyse the epidemiology and population structure of third-generation cephalosporin-resistant (3GCR) and carbapenem-resistant (CR) <i>Klebsiella pneumoniae</i> complex isolates, patients were screened for rectal colonisation with 3GCR/CR <i>K. pneumoniae</i> complex on admission to six German university hospitals (2016–2019). Also collected were 3GCR/CR and susceptible <i>K. pneumoniae</i> isolates from patients with bloodstream infections (2016–2018). Whole-genome sequencing was performed followed by multilocus sequencing typing (MLST), core-genome MLST, and resistome and virulome analysis. The admission prevalence of 3GCR <i>K. pneumoniae</i> complex isolates during the 4-year study period was 0.8%, and 1.0 bloodstream infection per 1000 patient admissions was caused by <i>K. pneumoniae</i> complex (3GCR prevalence, 15.1%). A total of seven <i>K. pneumoniae</i> complex bloodstream isolates were CR (0.8%). The majority of colonising and bloodstream 3GCR isolates were identified as <i>K. pneumoniae</i>, 96.7% and 98.8%, respectively; the remainder were <i>K. variicola</i> and <i>K. quasipneumoniae</i>. cgMLST showed a polyclonal population of colonising and bloodstream isolates, which was also reflected by MLST and virulome analysis. CTX-M-15 was the most prevalent extended-spectrum beta-lactamase, and 29.7% of the colonising and 48.8% of the bloodstream isolates were high-risk clones. The present study provides an insight into the polyclonal 3GCR <i>K. pneumoniae</i> population in German hospitals.
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