Listeria monocytogenes infection (listeriosis) is an uncommon but severe foodborne illness that affects mainly individuals with recognized underlying conditions: the elderly, immunocompromised individuals, and pregnant women and their fetuses. The aim of this study was to obtain epidemiological data on cases of listeriosis occurring in Portugal from 2008 through 2012, collected in hospitals on a voluntary basis. L. monocytogenes isolates were characterized by genoserotyping by multiplex polymerase chain reaction, DNA macrorestriction pulsed-field gel electrophoresis (PFGE), and determination of minimal inhibitory concentration (MIC, μg/mL) for 12 antibiotics. During this period, 203 cases of listeriosis were detected. The annual incidence rate observed ranged from 0.2 to 0.7 cases per 100,000 inhabitants. Nineteen cases (9.5%) corresponded to maternal/neonatal (MN) infections. The mean age of the nonmaternal/neonatal (non-MN) cases with documented age was 59 years, and 46.4% occurred in patients aged over 65 years. The majority of listeriosis cases were caused by genoserogroup IVb isolates, and PFGE analysis revealed a high molecular diversity, suggesting that most were sporadic. Nevertheless, several clusters of isolates presenting different geographic and time distributions were detected. The incidence of antibiotic-resistant isolates of L. monocytogenes was low but significantly higher than in previous years (2003-2007). The implementation of a national surveillance system monitoring the incidence of listeriosis and antimicrobial resistance of strains would be most valuable, allowing identification of sporadic and outbreak cases, to detect general trends in antibiotic susceptibilities, and potentially identify food sources of clinical strains.