Poultry products have been recognized as a major source of human illness caused by Salmonella serovars. The water content of pre-chiller and chiller in processing line could be the source of cross contamination. Water contents of pre-chillers and chillers, in Mashhad poultry abattoirs are regularly changed every 2 and 5 hours respectively. In this study, a total of 52 water samples were collected from the main tank, the pre-chiller, the initial and the final sections of the chiller in different hours and during four consecutive days. In order to isolate Salmonella spp., conventional culture method: including pre- enrichment, enrichment, selective plating and differential plating were performed. To confirm the identification of isolated colonies as Salmonella spp. and determining serovars as Typhimurium and Enteritidis serovars, a multiplex PCR (m-PCR) assay, using three pairs of primers were employed. S141 and S139 for InvA gene, specific for the genus of Salmonella, Fli15 and Tym for FliC gene, specific for Typhimurium serovar and Prot6e-5 and Prot6e-6 for Prot6E gene, specific for Enteritidis serovar. Number of ten samples (19.2 %) were determined as contaminated with Salmonella spp. All positive samples were from the source of water chiller and the highest rate of contamination was determined in the final hours before refilling of chiller water. In m-PCR assay, number of 1 (1.9%) of isolated colonies were confirmed as SalmonellaTyphimurium and number of 3 (5.8%) of isolated colonies were ...