Horizontally acquired genomic islands may allow bacteria to conquer and colonize previously uncharted niches. Four Klebsiella pneumoniae tRNA gene insertion hotspots (arg6, asn34, met56, and pheV) in 101 clinical isolates derived from blood, sputum, wound, bile or urine specimens were screened by long-range PCR for the presence or absence of integrated islands. The pheV phenylalanine tRNA gene was the most frequently occupied site and harbored at least three entirely distinct types of islands: (1) KpGI-1, a 3.7 kb island coding for four proteins, three of which showed high similarity to two hypothetical proteins and a Gcn5-related N-acetyltransferase in Salmonella enterica, (2) KpGI-2, a 6.4 kb island coding for five proteins including a truncated phage-like integrase, two helicase-related proteins, and a homolog of the functionally elusive Fic protein, and (3) KpGI-3, a 12.6 kb island which carried seven fimbriae-related genes, first identified in MGH78578. Consistent with the niche-adaptation hypothesis, KpGI-1-like islands which coded for the putative acetyltransferase were significantly over-represented in sputum isolates as compared to urine (P < 0.001), blood (P < 0.05) or bile (P < 0.05) derived isolates. Despite the unique nature of KpGI-2, likely homologs of orf5_KpGI-2 that coded for Fic were also found at undefined locations in six other clinical isolates, though none possessed the other KpGI-2 genes. We propose that the pheV-associated islands described in this study may contribute to fine tuning and adaptation of K. pneumoniae strains toward preferred infection and/or colonization pathways.