Shiga toxin-producingEscherichia coli(STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection ofstx₁,stx₂, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection ofstxand serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having completestxAandstxBsubunits. However, truncatedstxfragments occurred in twenty-two other isolates, some having multiplestxfragments in the genome. Isolates with completestxby WGS had consistentstx₁andstx₂detection by PCR, although one also having astx₂fragment had inconsistentstx₂PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates wereCitrobacterspp. Results demonstrate thatstxfragments associated withstx-carrying bacteriophages in theE.coligenome may contribute to inconsistent detection ofstx₁andstx₂by PCR. Fourteen isolates had integratedstxbacteriophage but lacked complete or fragmentarystxpossibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) wherestxwas located, likely increasing the stability ofstxin the bacterial genome and its detection by PCR.