In 2011, a Shiga toxin producing Enteroaggregative Escherichia coil (EAEC Stx2a+) 0104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolyticuremic syndrome (HUS) in Germany. In 2013, E. col O104:H4 isolates were obtained from a patient with HUS and her friend showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and compared. Whole-genome sequencing VGS) was performed for detailed characterization and to determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4 isolates of 2009 and 2011 available on NCB' were included in the virulence and phylogenetic analysis. All E. coil O104:H4 isolates tested were positive for stx2a, aatA, and terD but were negative for escV All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC Stx2a O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one allele difference was found between the isolate of the HUS-patient and that of her friend. Our study shows that EAEC Stx2a O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are still circulating necessitating proper surveillance to prevent further outbreaks with these potentially pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of 2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection control strategies.