<p>The objective of this study was to characterize the virulence characteristics of a collection of <i>Klebsiella pneumoniae</i> isolates collected from different clinical sources. A collection of 60 non-repetitive <i>K. pneumoniae</i> isolates, was studied. In vitro, virulence was analyzed by testing the survival of bacteria in pooled human serum. Isolates were typed by MLST. The genomes of 23 <i>K. pneumoniae</i> isolates, representatives of different STs and virulence profiles, were completely sequenced using the Illumina platform. Of note, 26/60 of <i>K. pneumoniae</i> isolates were resistant to killing by complement. Serum-resistant isolates belonged to distinct STs. Analysis of WGS data with VFDB showed the presence of several virulence genes related various virulence functions. Specifically, serum-resistant isolates carried a higher number of ORFs, which were associated with serum resistance, compared to serum-sensitive isolates. Additionally, analysis of WGS data showed the presence of multiple plasmid replicons that could be involved with the spread and acquisition of resistance and virulence genes. In conclusion, analysis of virulence characteristics showed that an important percentage (31.6%) of <i>K. pneumoniae</i> isolates were in vitro virulent by exhibiting resistance to serum. Thus, the presence of several virulence factors, in combination with the presence of multidrug resistance, could challenge antimicrobial therapy of infections caused by such bacteria.</p>