| Summary: | Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Consumption of chicken meat is considered the main route for human infection with <i>Campylobacter</i>. This study aimed to determine the critical factors for <i>Campylobacter</i> cross-contamination in Chinese commercial kitchens during chicken handling. Five commercial kitchens were visited to detect <i>Campylobacter</i> occurrence from 2019 to 2020. Chicken samples (n = 363) and cotton balls from the kitchen surfaces (n = 479) were collected, and total bacterial counts and <i>Campylobacter</i> spp. were detected. Genotypic characterization of 57 <i>C</i><i>ampylobacter jejuni</i> isolates was performed by multilocus sequence typing (MLST). In total, 77.41% of chicken carcass samples and 37.37% of kitchen surfaces showed <i>Campylobacter</i> spp. contamination. Before chicken preparation, <i>Campylobacter</i> spp. were already present in the kitchen environment; however, chicken handling significantly increased <i>Campylobacter</i> spp. prevalence (<i>p <</i> 0.05). After cleaning, boards, hands, and knives still showed high bacterial loads including <i>Campylobacter</i> spp., which related to poor sanitary conditions and ineffective handling practices. Poor sanitation conditions on kitchen surfaces offer greater opportunities for <i>Campylobacter</i> transmission. Molecular typing by MLST revealed that <i>Campylobacter</i> cross-contamination occurred during chicken preparation. The most prevalent sequence types, ST693 and ST45, showed strong biofilm formation ability. Consequently, sanitary condition of surfaces and biofilm formation ability of isolates were the critical points contributing to spread of <i>Campylobacter</i> in kitchen environment. These results provide insight into potential targeted control strategies along the farm-to-plate chain and highlight the necessity for improvements in sanitary conditions. The implementation of more effective cleaning measures should be considered to decrease the campylobacteriosis risk.
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