Aim To study the prevalence and distribution of various variants in the stx gene of Shiga toxinproducing Escherichia coli (STEC) isolated from diverse environmental sources (animal stool, meat) and human illness, from a large geographic area in India, and to understand the association between variants, serotype distribution and human disease. Methods and Results A surveillance for STEC was conducted in the semi-urban and rural areas of Punjab, Himachal, Haryana and Chandigarh. Shiga toxinproducing Escherichia coli isolates (80 animal stool, 39 meat, 21 human stool from diarrhoea and HUS cases) were characterized for stx variants by PCR. Shiga-like toxin (Stx) was detected using Ridascreen-EIA assay. Variant stx2c was the most common (25.1%), followed by stx1d (13%), stx1c (10.7%) and stx2d (9.2%), whereas stx2e, stx2f and stx2g were absent. Only 8/21 (38%) human isolates harboured stx variants, of which stx2c and stx2d were found in 2 and 1 isolates, respectively. The low frequency of carriage of these potentially more pathogenic variants may explain the low severity of human illness seen in India. Shiga-like toxin was detected in only 42 of the isolates positive for the stx genes probably due to the low levels of toxins produced. Serogroup distribution was found to be diverse, suggesting the lack of any predominant circulating type. Conclusions The presence of stx variants 1c, 1d, 2c and stx2d in diverse environmental and human sources in India was demonstrated. The prevalence of the most common subtype stx2c found in this study in animal isolates may pose a threat to the public health. We report the subtyping of human STEC isolates and report the presence of stx1d subtype for the first time from India. Significance and Impact of the Study We demonstrated the presence of potentially pathogenic subtypes in the environmental specimens which may act as a reservoir for human infections. Serogroups new to India were also reported.