Escherichia coli serotype O157:H7 was first recognized in 1982 as a human pathogen associated with outbreaks of bloody diarrhea in the United States and is now considered a major cause of foodborne infections because of its high hospitalization rate. Cattle is the major reservoir of E. coli O157:H7. Cattle harbor E. coli O157:H7 in the hindgut and shed the organisms in the feces, which serves as a source of contamination of food and water. It is hypothesized that dietary ingredients that reach the hindgut are likely to affect colonization and fecal shedding of STEC. Increased flow of dietary ingredients (starch, fiber, protein, and lipid) are likely to alter ecology of the hindgut, resulting in altered pH and fermentation products, which could have a positive or negative impact on E. coli O157:H7. The objectives of this study are to investigate E. coli O157:H7 populations in fecal shedding of cattle. The cattle in this study were fed with diets with different levels of ruminally-degradable and –undegradable protein. A total of 286 E. coli O157:H7 isolates were recovered from feces of 576 crossbred calves at the Clayton Livestock Research Center in Clayton, New Mexico. The organisms were sequenced using Illumina Miseq system. De novo assembly of raw reads was performed using SPAdes and SNPs analysis of the isolates was conducted using kSNP3. Virulence factor Database (VFDB), created by the MOH key laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, was used as a reference for BLAST. The results indicated that increased flow of undegradable protein may increase the shedding of E. coli O157. However, the effect of ractopamine was still unknown. Three clades were identified among the E. coli O157:H7 isolates, including clades 6, 7, and 8, most of which belong to clade 8 (205 of 286). 49588 SNPs were found according to kSNP3. 19043 SNPs were identified as core SNPs. The phylogenetic analysis showed that the E. coli O157 isolates which collected from neighboring Pens were more closely to each other.