The objective of this study was to assess the genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates from cattle. Multi-locus sequence typing (MLST) was used to identify and compare the sequence types (STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curated by the STEC Center at Michigan State University. For the 43 isolates, 19 STs were identified and 10 of those STs were novel compared to those in EcMLST. For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28 and O8:H19 were most common, and STEC O8 isolates were the most diverse, with seven different STs for isolates with that O group. STEC strains with O types identified in this study have been isolated from cattle by other researchers, as well as from cases of human gastroenteritis. Of the 10 novel STs identified, six were found to be closely related to previously identified STs, indicating that populations of non-O157 STEC in cattle are similar to those from other sources, including human clinical cases. Significance and impact of the study: The foodborne pathogen Shiga toxin-producing Escherichia coli (STEC) is a significant public health concern. One of the main reservoirs for STEC are cattle, which can directly or indirectly contribute to STEC in the food supply. The genetic subtype data presented here highlight the diversity of STEC that can be isolated from cattle. These results further our understanding of the ecology of STEC in the primary production environment, which is important for developing effective control measures to reduce this pathogen in the food supply.