In order to describe the genomic characteristics related to antimicrobial resistance and comparative genomics of diarrheagenic Escherichia coli (DEC), 14 DEC isolates from the strain collection of the Instituto Nacional de Salud (INS) were subjected to genome sequencing. We used bioinformatic procedures to analyze the obtained sequences in order to look for microbial resistance genes and genetic regions related to pathotypes and phylogroups. Several antimicrobial resistance determinants were detected, but the production of beta-lactamases and mutations associated to quinolone resistance were the most relevant. Additionally, we observed isolates of the same pathotype grouped in different phylogroups. The comparative genomics analysis showed a greater number of orthologous genes in isolates from the same pathotype and phylogroup. In conclusion, DEC isolates from Lima, Peru, showed resistance to multiple drugs; likewise, molecular and phylogenetic diversity was observed in several pathotypes and phylogroups.