The combination of the traditional culture-dependent method and high-throughput sequencing was used to investigate the level of microbial contamination on the surface of pig carcasses during the slaughter and segmentation process. Meanwhile, the number of microbial colonies on the slaughter knives and the contact surfaces of the segmentation workshop were counted to identify the key pollution links in the slaughter and segmentation process. The results showed that a total of 881458 valid sequences and 864 operational taxonomic units (OTUs) were obtained by sequencing. The samples were annotated to 22 phyla, 33 classes, 79 orders, 162 families, 382 genera and 613 species of microorganisms. Proteobacteria, Bacteroidota and Firmicutes were the dominant bacterial phyla. Acinetobacter and Aeromonas were the major dominant bacterial genus. The bacterial community diversity during the slaughter and segmentation process was in decreasing order as follows: bleeding > dehairing > segmentation > evisceration > final wash > chilling. The microbial diversity on the carcass surface was the lowest in the chilling stage, and increased after segmentation, indicating that the segmentation stage was the key contamination link. The results of traditional microbial counting were consistent with the results of sequencing. From dehairing to chilling, the number of each bacterial group on the surface of pig carcasses was decreased, but increased significantly after segmentation. The total number of bacterial colonies on the carcass surfaces in the segmentation workshop was 6.11 (lg(CFU/cm²)) on average, which was higher than that on the slaughter knives (4.86 (lg(CFU/cm²)) on average), indicating that the contact surfaces of the segmentation workshop were the key pollution source, and so the segmentation link was the key pollution link.