Vast antibiotic concentration gradients exist in the environment and within humans undergoing treatment. Previous studies in the area focus primarily on antibiotic concentrations well above lethal levels, leaving characteristics of populations present in sub-lethal concentrations a missing piece in our knowledge of resistance development. Here we aim to holistically investigate the role of sub-lethal antibiotic concentrations in the evolution of antibiotic resistance in the context of different environment structures and elucidate underlying molecular mechanisms. We exposed E. coli to various sub-lethal ciprofloxacin concentrations in either swim agar or well mixed liquid culture environments over 3 days and monitored resistance development with minimum inhibitory concentration assays. We combined a single-cell microfluidics technique with time-lapse microscopy, and whole genome sequencing to find both sub-lethal antibiotic concentrations, and environment structure, have a significant impact in the development of antibiotic resistance mechanisms. Well mixed cultures showed significant mutations in genes associated with transcriptional repression and DNA gyrase, key controllers of AMR. Swim agar cultured mutants also showed mutations in transcriptional repression in addition to genes involved in regulation of efflux pumps. We then link these mutations to phenotypic heterogeneity in cell growth, with the different resistant mutants having different growth dynamics during and after a fresh antibiotic challenge. These results highlight for the first time the importance that different growth environments have in AMR development. We show the different resistance mechanisms that develop under different antibiotic concentrations and environment structures, both of which are vital if we are to identify, tackle and overcome AMR.