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House flies (Musca domestica L. [Diptera: Muscidae]) can act as a mechanical vector for food-borne pathogens including Shiga toxin-producing Escherichia coli (Migula; Enterobacteriales: Enterobacteriaceae) (STEC) in and around cattle feedlots.The present study assessed the prevalence of STEC in house flies from a restaurant area of a town in northeastern Kansas.Two hundred twenty-four house flies were collected over 10 wk, surface sterilized, individually homogenized, and cultured by a multifaceted approach of direct plating on selective media and an enrichment broth, followed by the immunomagnetic separation. Bacterial isolates were screened for eight serogroups of E. coli: O103, O104, O26, O111, O45, O145, O121, and O157 using multiplex polymerase chain reaction (PCR). Furthermore, O-serogroup-positive isolates were tested for virulence genes stx1, stx2, eae, and ehxA by PCR.The majority (91.5%) of flies carried enteric bacteria, and the mean value of enteric concentration on the modified Possé agar was 6.7 ± 1.1 × 106 colony forming units per fly. Thirty-nine of the 224 flies (17.4%) were positive for one or more E. coli serogroup of interest; with the majority O103 (10.7%), followed by O26 (3.1%), O121 (1.3%), O45 (1.3%), and O104 (0.9%). However, none of the serogroup-positive isolates carried any of the virulence genes tested. Results of our study show that house flies in the urban environment do not carry STEC. Nevertheless, detection of E. coli O-serogroups with the potential to acquire virulence traits indicates that house flies in an urban environment represent a public health risk.