Antibiotic resistance is a global health threat and consequently, there is a need to understand the mechanisms driving its emergence. Here, we hypothesize that genes and mutations under positive selection may contribute to antibiotic resistance. We explored wastewater E. coli, whose genomes are highly diverse. We subjected 92 genomes to a statistical analysis for positively selected genes. We obtained 75 genes under positive selection and explored their potential for antibiotic resistance. We found that eight genes have functions relating to antibiotic resistance, such as biofilm formation, membrane permeability, and bacterial persistence. Finally, we correlated the presence/absence of non-synonymous mutations in positively selected sites of the genes with a function in resistance against 20 most prescribed antibiotics. We identified mutations associated with antibiotic resistance in two genes: the porin ompC and the bacterial persistence gene hipA. These mutations are located at the surface of the proteins and may hence have a direct effect on structure and function. For hipA, we hypothesize that the mutations influence its interaction with hipB and that they enhance the capacity for dormancy as a strategy to evade antibiotics. Overall, genomic data and positive selection analyses uncover novel insights into mechanisms driving antibiotic resistance.