ABSTRACT Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically significant food-borne pathogens. However, there is a dearth of information on serotype prevalence and virulence gene distribution, data essential for the development of public health protection monitoring and control activities for the meat and dairy industries. Thus, the objective of this study was to examine the prevalence of non-O157 STEC on beef and dairy farms and to characterize the isolates in terms of serotype and virulence markers. Bovine fecal samples ( n = 1,200) and farm soil samples ( n = 600) were collected from 20 farms throughout Ireland over a 12-month period. Shiga toxin-positive samples were cultured and colonies examined for the presence of stx ₁ and/or stx ₂ genes by PCR. Positive isolates were serotyped and examined for a range of virulence factors, including eaeA , hlyA , tir , espA , espB , katP , espP , etpD , saa , sab , toxB , iha , lpfA _O157/OI-141 , lpfA _O113 , and lpfA _O157/OI-154 . Shiga toxin and intimin genes were further examined for known variants. Significant numbers of fecal (40%) and soil (27%) samples were stx positive, with a surge observed in late summer-early autumn. One hundred seven STEC isolates were recovered, representing 17 serotypes. O26:H11 and O145:H28 were the most clinically significant, with O113:H4 being the most frequently isolated. However, O2:H27, O13/O15:H2, and ONT:H27 also carried stx ₁ and/or stx ₂ and eaeA and may be emerging pathogens.