This treatise was conducted to investigate Listeria presence in one cold-smoked salmon (CSS) processing factory, Al-Sharkia Governorate, Egypt as well as to evaluate detection accuracy of Agar Listeria according to Ottaviani and Agosti (ALOA) using 16S rRNA technique. A total of 50 and 60 samples from final products and their processing line six-point (PLS) have been tested for the occurrence of this bacterium, respectively. The above technique showed that Bacillus safensis, Oceanobacillus picturae and B. wiedmannii have been found in CSS. The presumptive of Listeria spp. (L. monocytogens) in CSS and PLS were found to be 8 and 16.67%, respectively while presumptive of Listeria spp. (L. innocua) were 26 and 16.67% respectively. The high occurrence percentage of this pathogen in PLS of factory could be attributed to cross-contamination. As a direct consequence, Listeria spp. control must be implemented through a proper cleaning and sanitation programme as well as a HACCP plan to prevent cross-contamination and spread of these pathogens to final products during the fishery processing.