Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relatively infrequent, their broad scope, regulatory importance, and severe health outcomes highlight the need to better understand the environmental survival of manure-borne pathogens. Shiga toxigenic Escherichia coli (STEC) are excreted in feces and persist in the environment until they die or recolonize a new host. Surface waters contaminated with manure-borne STEC can infect humans through drinking and recreational water use or irrigated crops that are minimally cooked. In this study, manure-impacted water microcosms mimicking beef cattle feedlot runoff were used to assess survival of STEC strains representing seven STEC serotypes (O26, O45, O103, O111, O121, O145, and O157) and persistence of target O antigen genes. Microcosms were sampled over the course of 1 year, and the entire experiment was repeated in a second year. Culture and polymerase chain reaction (PCR)-based techniques were used for detection and enumeration. Serotype-specific survival results were observed. Both STEC O26 and O45 declined slowly and remained culturable at 24 months. In contrast, STEC O121 and O145 decreased rapidly (-0.84 and -1.99 log10 abundance per month, respectively) and were unculturable by months 4 and 5, but detectable by PCR for a mean of 4.5 and 8.3 months, respectively. STEC O103, O111, and O157 remained culturable for a mean of 11.6, 5.5, and 15 months and detectable by PCR for a mean of 12, 13.8, and 18.6 months after inoculation, respectively. Results document that some STEC serotypes have the biological potential to survive in manure-impacted waters for extended periods of time when competing microflora are eliminated. Serotype-specific differences in survival of target bacteria and persistence of target genes were observed in this sample set, with STEC O26 and O45 strains appearing the most robust in these microcosm studies.