To better understand the epizootiology of Escherichia coli O157:H7 among cattle, all E. coli O157 isolates recovered on a research feedlot during a single feeding period were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Three distinct MLVA subtypes (A, B, C), accounting for 24%, 15%, and 64% of total isolates, respectively, were identified. Subtypes A and B were isolated at the initiation of sampling, but their prevalence waned and subtype C, first isolated on the third sampling date, became the predominant subtype on the feedlot. Supershedding events, however, occurred with equal frequency for all three MLVA-types. Using a multilevel logistic regression model, we investigated whether the odds of shedding subtype C relative to subtypes A or B were associated with time, diet, or the presence of a penmate shedding high numbers of subtype C. Only time and exposure to an animal shedding MLVA-type C at 10³ colony-forming units or greater in the pen at the time of sampling were significantly associated with increased shedding of subtype C. High-level shedding of those E. coli O157 subtypes better suited for survival in the environment and/or in the host appear to play a significant role in the development of predominant E. coli O157 subtypes. Supershedding events alone are neither required nor sufficient to drive the epidemiology of specific E. coli O157 subtypes. Additional factors are necessary to direct successful on-farm transmission of E. coli O157.