The prevailing global spread of four dengue virus (DENV) serotypes and the resultant co-circulation of multiple serotypes in the same region have invariably led to conditions supporting the periodic occurrence of simultaneous infection of individuals with more than one DENV serotype. This raises the issue of how best to detect concurrent multiple infections. We report here the use of a nested reverse transcription-polymerase chain reaction (RT-PCR) assay, which detected concurrent infection with three DENV serotypes (DENV-1/DENV-2/DENV-3) and two serotypes (DENV-1/DENV-2 and DENV-2/DENV-4), respectively, in three serum specimens from Thai children hospitalized during the dengue epidemic of 2000-2001. In contrast, an enzyme-linked immunosorbent assay used previously for virus serotype identification failed to detect multiple DENV serotypes in these specimens. Serotype identification by RT-PCR was confirmed by sequence analysis of each amplified PCR product. Phylogenetic analyses performed on PCR-amplified DNA fragments further supported the occurrence of concurrent infections with multiple DENV serotypes in these children. Although the sample set was small, our data suggest that nested RT-PCR is an effective method for the detection of concurrent DENV infections.