The information about disease burden and epidemiology of invasive listeriosis in Asia is scarce. From 2000 to 2013, a total of 338 patients with invasive listeriosis (bacteremia, meningitis, and peritonitis) were treated at four medical centers in Taiwan. The incidence (per 10,000 admissions) of invasive listeriosis increased significantly during the 14-year period among the four centers (0.15 in 2000 and >1.25 during 2010-2012) and at each of the four medical centers. Among these patients, 45.9% were elderly (>65 years old) and 3.3% were less than one year of age. More than one-third (36.7%) of the patients acquired invasive listeriosis in the spring (April to June). Among the 132 preserved Listeria monocytogenes isolates analyzed, the most frequently isolated PCR serogroup-sequence type (ST) was IIb-ST87 (23.5%), followed by IIa-ST378 (19.7%) and IIa-ST155 (12.1%). Isolation of PCR serogroups IIb and IVb increased significantly with year, with a predominance of IIb-ST87 isolates (23.5%) and IIb-ST 228 isolates emerging in 2013. A total of 12 different randomly amplified polymorphic DNA (RAPD) patterns (Patterns I to XII) were identified among the 112 L. monocytogenes isolates belonging to eight main PCR serogroup-STs. Identical RAPD patterns were found among the isolates exhibiting the same PCR serogroup-ST. In conclusion, our study revealed that during 2000-2013, listeriosis at four medical centers in Taiwan was caused by heterogeneous strains and that the upsurge in incidence beginning in 2005 was caused by at least two predominant clones.