Australian beef processors must ensure that products exported to the US market are deemed free of regulatory important serotypes of Shiga toxin-producing Escherichia coli (STEC). Better understanding of the microbiota and its movement in the abattoir could assist in determining the sites of cross-contamination of top 7 STEC serotypes (O26, O45, O103, O111, O121, O145 and O157) through slaughter. This study aimed to investigate changes in microbial populations and the top 7 STEC during slaughter in two Australian beef export abattoirs with different supply chains. Abattoir A (integrated) and B (fragmented) were visited twice and once, respectively. At each visit 90 samples consisting of 10 faecal samples from the holding pens, 15 from hides, 15 from post-hide pull carcases, 15 from post-evisceration carcases, 15 from pre-chill carcases and 10 environmental samples were collected. Samples were assessed for total viable count (TVC), E. coli/coliforms, and traditional (stx, eae and O-antigens) and novel (espK and espV) STEC markers. Culture confirmation was conducted on potential positive (PP) samples (stx+, eae+ and O-antigen+). Resulting isolates were characterised for traditional and novel STEC markers. TVC of hide samples and pre-chill carcases from abattoir A were significantly higher (P<0.05) than abattoir B with an average difference of 0.88 and 0.39 log10/cm2, respectively. TVC of post hide pull samples from abattoir B was significantly higher (P<0.05) than abattoir A with an average difference of 0.7 log10/cm2. There were no differences between TVC counts for other samples. E. coli were present in all hide and faecal samples but were significantly higher (P<0.05) in samples from abattoir A than abattoir B. E. coli was intermittently present and significantly higher (P<0.05) in carcases samples from abattoir B than abattoir A. PP’s were identified in 62, 58 (abattoir A) and 58 (abattoir B) samples. PP’s decreased to 48, 43 and 36 samples, respectively, with the addition of the novel markers. STEC isolates were only recovered from hide and faecal samples with three O157 and two O111 recovered from all samples tested. Further metagenomics studies will assist in understanding how microbial populations vary through slaughter.