Dengue virus serotype 3 (dengue-3) has been classified into five genotypes (I-V) by phylogenetic analysis based on different viral genes. To investigate the genetic variability and evolutionary character of the dengue-3 isolates in southern Taiwan from 2005 to 2006, we analyzed the 290 nucleotides of the core (C) gene of 12 dengue-3 isolates and compared them with the published C gene sequences of global dengue-3 strains available in GenBank, including four isolates from 1998 and one isolate from 1999, from Taiwan. The dengue-3 viruses from 2005 to 2006 were not from continuous spread of an epidemic strain or re-emergence of the 2005 strains in the 2-year period because there was a 5.4-6.2% difference in the 290 nucleotides of the C gene and different genotypes between the 2005 and 2006 strains. Most of the nucleotide changes, compared with a prototype dengue-3 virus, H87, occurred in the third codon position and were non-synonymous mutations occurring naturally in the C gene. In addition, there was no consistent difference in the 290 nucleotides of the C gene between eight dengue fever and two dengue hemorrhagic fever isolates from 2006. The phylogenetic analysis indicates that the isolates from the 1998, 1999 and 2006 Taiwan dengue-3 epidemics are phylogenetically related and belong to genotype III. It was noted that the 2005 Taiwan dengue-3 isolates belong to another genotype. This molecular epidemiology study of dengue-3 viruses in Taiwan helps to elucidate whether there is a continuation of outbreaks in consecutive years, re-emergence of endemic dengue virus, or introduction of strains from other countries.