The aims of this study were to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) in bulk tank ewes’ milk and farm environment in Castilla y León (Northwest Spain) and to characterize the isolates obtained to gain information on reservoirs and sources of contamination.Milk samples from 388 dairy farms were examined by immunoassay, culture methods and PCR. Additional samples from air, water, feed and faeces from 10 farms were analyzed by culture methods and PCR. All the samples of bulk tank milk tested with the immunoassay yielded negative results. By PCR, stx genes were detected in 176 out of 388 (45.4%) milk samples, 9 out of 20 (45%) water samples and 5 out of 20 (25%) faecal samples. A total of 47 isolates were obtained, four of them belonging to serotype O157:H7 (three from milk samples and one from water samples).In conclusion, Spanish bulk tank ewes’ milk can constitute a source of STEC, but the overall prevalence of serotypes involved in human disease is low. The environment of the dairy farm may represent an important reservoir of this microorganism.