A Danish pig slaughterhouse was visited in this study to investigate the impact of carcass processing on prevalence of tetracycline-resistant Escherichia coli, and to identify the origins of carcass contaminations with E. coli by assessing genetic diversity of E. coli populations on carcasses. A total of 105 carcasses were sampled at five sequential stages: after stunning, after scalding, after splitting, after cooling and after cutting. Total and tetracycline-resistant E. coli were counted for each sample and tetracycline resistance prevalence per sample was calculated by the fraction of tetracycline-resistant E. coli out of total E. coli. From 15 repeatedly sampled carcasses, 422 E. coli isolates from faeces, stunned carcasses, split carcasses and chilled carcasses were examined by pulse-field gel electrophoresis (PFGE) and tested for antimicrobial susceptibility. The results showed that E. coli counts and the prevalence of tetracycline-resistant E. coli per sample were both progressively reduced after each sampling stage. PFGE analysis showed that E. coli populations from stunned carcasses were highly genetically diverse, compared with those from split carcasses and faeces. Thirteen carcasses (87%) were contaminated with E. coli that were also isolated from faeces of either the same or other pigs slaughtered on the same day; and 80% of stunned carcasses shared the same E. coli PFGE subtypes. The results suggest that some carcass processing steps in the slaughterhouse were effective in reducing both E. coli numbers and the tetracycline resistance prevalence in E. coli on carcasses. Faeces from the same or other pigs slaughtered on the same day were likely to be an important source of E. coli carcass contamination. Combined data from E. coli enumeration, PFGE typing and antimicrobial susceptibility testing suggest that tetracycline-susceptible E. coli probably persisted better compared to resistant ones during the carcass processing.