Increasing emergence of salmonellosis presents a threat to the effective control of foodborne disease in humans. The purpose of this study was to evaluate the prevalence of drug susceptibility and molecular characteristics of non-typhoidal Salmonella (NTS) isolated from laying hens (LH) in 3 Moroccan regions, Rabat-Salé-Zemmour-Zaër (RSZZ), Souss-Massa-Drâa (SMD), and the grand Casablanca (GC). A total of 351 samples were collected from 30 consumer egg laying houses at the end of the egg laying period from April to July 2011. Sixty-four out of these 351 examined samples were contaminated by Salmonella. The Salmonella isolated strains were then serotyped and tested for drug susceptibility and analyzed by polymerase chain reaction (PCR) for the presence of the invasion-associated genes invA and spvC and nalidixic acid resistance-associated qnr gene. The prevalence of NTS infection in LH was estimated to be 73.3%. Seven Salmonella enterica serovars were identified: Enteritidis (37.5%), Kentucky (31.2%), Infantis (10.9%), Typhimurium (6.2%), Thompson (6.2%), Agona (4.6%), and Amsterdam (3.1%). Drug susceptibility testing showed that 65.6% of Salmonella were resistant to at least one antibiotic and 25% were resistant to ciprofloxacin. All isolates were positive for the invasion gene invA and 28% of them were positive for the virulence gene spvC. All nalidixic acid-resistant S. Enteritidis isolates were negative for qnr plasmid genes. Our findings clearly suggest the necessity to establish an NTS monitoring and control program for LH in Morocco.