Aims: The purpose of this study was to investigate the occurrence of E. coli O157 and O26 on Belgian dairy cattle farms, the presence of virulence genes in the confirmed isolates, and the association of E. coli O26 presence with calf diarrhea. Methods and Results: In total, 233 recto‐anal mucosal swabs (RAMS) were obtained from healthy and diarrheic dairy calves on three farms, each alternately visited three consecutive times. RAMS were analyzed for presence of E. coli O157 and O26, and stx1, stx2 and eae virulence genes. Overall, 19% of RAMS tested positive for E. coli O157, while 31% tested positive for E. coli O26. The majority of isolates possessed both stx and eae, denoting a high pathogenic potential to humans. While both serogroups persisted at farm level, persistence within the same animal over time appeared to be relatively rare. Interestingly, E. coli O26 was already abundantly present at a younger age compared to E. coli O157. Calf diarrhea could not be associated with presence of E. coli O26. Conclusions: Young dairy calves are important on‐farm reservoirs of potentially pathogenic E. coli O157 and O26. A role of E. coli O26 in calf diarrhea could not be confirmed. Significance and Impact of Study: O157 and O26 are responsible for the majority of human STEC infections. Gaining more epidemiological information regarding their occurrence and persistence on cattle farms will contribute to a better understanding of STEC ecology and risk of human transmission.