This study aimed to understand the impact on virulence and fitness of mutations in specific genes found after adaptation of Klebsiella pneumoniae to colistin. Isolates with an increase in their inhibitory concentration (MIC) to colistin of 32- to >128-fold were shown to have mutations in mgrB, phoPQ and pmrAB, all known regulators of pathways affecting membrane lipid content. When these strains were used in studies in Galleria mellonella there was no clear correlation between mutations in specific genes per se and loss of virulence. Strains which showed sequence duplication in the HAMP-domain of PmrB showed reduced virulence but strains with point mutations in pmrAB showed no decrease in virulence. Similarly, specific mutations in mgrB in individual strains showed either loss of virulence or no effect/increased virulence. This study suggests that the impact on virulence may be independent of the colistin resistance mechanism and reflects differences in individual strain backgrounds.