Colistin, a crucial last-line antibiotic, is often used to combat multidrug-resistant bacteria in humans and animals. However, clinical bacterial isolates frequently exhibit mixed populations where a predominant susceptible variant masks the existence of low-frequency and unstable colistin heteroresistant (HR) cells. However, the mechanism for the emergence of such variants and the establishment of permanent resistance to colistin is not well understood. Using a combination of a quantitative Resistance Emergence Assay (REA), single cell imaging, sequencing, and mathematical modeling, we demonstrate that bacteria tolerant to colistin can spontaneously emerge within a few generations even when exposed to concentrations that exceed the MIC. Further, we demonstrate that these low-frequency tolerant bacteria lead to the emergence of heteroresistance, contrary to prior postulates that attributed this to pre-existing strains. Early tolerant cells are dominated by altered transcriptional activity and gene duplications, albeit at the cost of fitness defects. Hence, these modifications are unstable and quickly lost in the absence of colistin. However, the occasional acquisition of site-specific point allows the bacteria to rid themselves of the gene duplications while fixing the resistance in the population. Colistin HR mutant strains and their tolerant precursors exhibit a characteristic modification of the lipid membrane environment that affects the binding and efficacy of colistin.  Overall, our study reveals that phenotypic tolerance, rapid gene duplication, and resistance fixation by site-specific mutations, rather than pre-existing resistant subpopulations, is a common pathway for the emergence of colistin resistance, highlighting the need to target transient bacterial intermediates to improve antibiotic treatment strategies.