Escherichia coli is the main bacterial cause of major economic losses and animal welfare issues in poultry production. In this study, we investigate the effect of an autogenous vaccine on E. coli strains longitudinally isolated from broiler breeder flocks on two farms. In total, 115 E. coli isolates were sequenced using Illumina technologies, and compared based on a single-nucleotide pol-ymorphism (SNP) analysis of the core-genome and antimicrobial resistance (AMR) genes they car-ried. The results showed that SNP-based phylogeny corresponds to a previous multilocus-sequence typing (MLST)-based phylogeny. Highly virulent sequence types (STs), including ST117-F, ST95-B2, ST131-B2 and ST390-B2, showed a higher level of homogeneity. On the other hand, less frequent STs, such as ST1485, ST3232, ST7013 and ST8573, were phylogenetically more distant and carried a higher number of antimicrobial resistance genes in most cases. In total, 25 antimicrobial genes were detected, of which the most prevalent were mdf(A) (100%), sitABCD (71.3%) and tet(A) (13.91%). The frequency of AMR genes showed a decreasing trend over time in both farms. The highest prevalence was detected in strains belonging to the B1 phylogenetic group, confirming the previous notion that commensal strains act as reservoirs and carry more resistance genes than pathogenic strains that are mostly associated with virulence genes.