Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes severe illness in humans and is an important cause of foodborne disease. In Chile, there is limited information on the virulence characteristics of this pathogen in livestock, and none in companion animals. The aim of this study was to characterize STEC strains isolated from cattle, swine, dogs, and cats, in Chile, in terms of the presence of Shiga toxin types and subtypes, virulence genes, serogroups, and clonality. One-thousand two-hundred samples were collected, isolating 54 strains (4.5%), where stx1a (68.5%) and ehxA (74.1%) were the most frequently detected virulence genes. Only one strain belonging to the most clinically relevant serogroups was identified. Pulsed field gel electrophoresis analysis showed high clonal diversity among strains isolated from cattle, while those from swine showed the same pattern. This study provides further evidence regarding cattle and swine in Chile as a potential source of a wide variety of STEC strains that could potentially cause severe illness in humans, and that companion animals do not seem to represent a relevant reservoir. It also argues that preventive and control strategies should not be focused on detecting serogroups, but instead, on detecting their determinants of virulence.