The present study was designed to investigate the prevalence of Salmonella species, their molecular characterization and antibiotic resistance in backyard chickens. A total of 1125 samples were collected from backyard chickens in two consequent samplings. In the first part, samples were included of 820 poor recently hatched chicks, hatching residuals, egg shells in the nest floor, cloacal swabs and fresh litter droppings in the villages which located in north of Iran. Secondly, 305 samples were taken from newly hatched-chicks which fertile eggs were obtained from the rural chickens of those regions and incubated in laboratory incubator. Of 1125 samples tested, 27 (2.4 %) Salmonella were isolated that identified as serovars of Salmonella enteritidis (55.5 %), Salmonella typhimurium (22.2 %), Salmonella hadar (14.8 %) and Salmonella infantis (7.4 %). Except the traditional serotyping that was performed for all isolates, Salmonella typhimurium and Salmonella enteritidis isolates were characterized by using multiplex PCR for further identification. All of six Typhimurium serovars were positive for rfbJ, fljB, invA and fliC genes. In the case of Enteritidis serovars, polymerase chain reaction generated amplification products for spv, sefA and random sequence (specific for the genus Salmonella) in all of fifteen samples. Most of the Salmonella isolates in this study were sensitive to norfloxacin.