ABSTRACT Following two O121 Shiga toxin–producing Escherichia coli (STEC) outbreaks linked to wheat flour, this study was conducted to gain baseline information on the occurrence of bacterial pathogens and levels of indicator organisms in wheat flour in Canada. A total of 347 prepackaged wheat flour samples were analyzed for Salmonella species, STEC, Listeria monocytogenes, aerobic colony count (ACC), total coliforms, and Escherichia coli . Salmonella spp. and O157 STEC were not detected in any of the samples. L. monocytogenes was identified in two samples (0.6%) at levels below the limit of detection (<0.7 log CFU/g). Non-O157 STEC were isolated from six samples (1.7%) and were characterized for the presence of STEC virulence genes: stx 1 , stx 2 , and their subtypes, eae, hlyA, and aggR . One O103:H25 STEC isolate carried virulence genes ( stx 1a + eae ) that are known to be capable of causing diarrhea and/or bloody diarrhea in humans. Of the five remaining non-O157 STEC isolates, four carried single stx 2a or stx 2c genes and were considered to have the potential of causing diarrhea. The remaining non-O157 STEC isolate ( stx 2 ), while not a priority non-O157 STEC, was not available for sequencing; thus, its potential to cause illness is unknown. ACC, total coliforms, and E. coli were detected (≥0.48 log CFU/g) in 98.8, 72.6, and 0.6% of the flour samples. The mean counts of ACC were greater in whole wheat flour compared with the other flour types tested ( P < 0.001). The results of this study suggest that the occurrence of O157 STEC and Salmonella is low but that the occurrence of non-O157 STEC in wheat flour with the potential to cause human illness of diarrhea is relatively common. Therefore, the consumption of raw flour could increase the likelihood of STEC infections. Further research is merited for potential risk mitigation strategies within the food production system and with consumers. HIGHLIGHTS O157 STEC and Salmonella were not found. Non-O157 STEC was found in six samples (1.7%), of which five carried the stx gene only. One non-O157 STEC (O103:H25) isolate carried virulence genes stx 1a and eae . L. monocytogenes was identified in two samples (0.6%) below the detectable counts. ACC, coliforms, and E. coli were detected in 98.8, 72.6, and 0.6% of samples.