This study aimed to evaluate different detection methods in determining Salmonella prevalence in layers of various rearing periods, and to serotype, ribotype and antibiotype the isolates. For this, 174 drag swab samples taken from 58 commercial layer flocks of 15, 25, and 40 week age were analyzed for Salmonella by ISO standard culture method (ISO 6579:2002/Amd 1:2007 - ISO) and by 2 real time polymerase chain reaction (rPCR) systems (Light Cycler SYBR Green I rPCR - LCSGIrPCR and DuPont BAX Q7 rPCR - BAXrPCR). Seventeen out of 58 (29.31%) flock samples, and 6 of the 15th and 25th weeks' (3.44% and 3.44%), and 10 (5.74%) of the 40th week's drag swab samples were Salmonella positive by all 3 methods. Twenty out of 22 Salmonella isolates (90.90%) were Salmonella enterica subsp. enterica, including serovars of 9 Enteritidis (SE) (40.9%), 7 Infantis (SI) (31.8%), 1 Hadar (4.5%), 1 Montevideo (4.5%), 1 Colombo (4.5%), 1 Spartel (4.5%), and 2 subspecies (9.10%) as Salmonella enterica subsp. arizonae and Salmonella enterica subsp. houtenae. Also isolates showed resistance to 23 of 24 antibiotics, with the highest resistances to Ampicillin (100%), Neomycin (100%), Penicillin G (100%) and Erythromycin (95.45%). Results indicate that both LCSGIrPCR and BAXrPCR systems can be used to complement ISO method in rapid detection of Salmonella from layer chickens, and that there is high prevalence of Salmonella enterica subsp. enterica in all rearing periods examined.