Objective:To study evolutionary relationship of the 5’untranslated regions(5’UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution(1977-2000) comparing to the DEN3prototype(H87).Methods:The 5’UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced.Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software.Replication of five Thai DEN3 candidates comparing to the 1187 prototype were done in human(HepG2) and the mosquito(C6/36) cell lines.Results:Among these Thai DEN3,the completely identical sequences of their first 89 nucleotides,their high-order secondary structure of 5’UTRs and three SNPs including the predominant C90 T,and two minor SNPs including A109 G and A112 G were found.The C90 T of Thai DEN3.Bangkok isolates was shown predominantly before 1977.Five Thai DEN3 candidates with the predominant C90 T were shown to replicate in human(HepG2) and the mosquito(C6/36) cell lines better than the H87 prototype.However,their highly conserved sequences as well as SNPs of the 5’UTR did not appear to correlate with their disease severity in human.Conclusions:Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence,the high-order secondary structure and the predominant C90 T of the 5’UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3. Objective: To study evolutionary relationship of the 5untranslated regions (5’UTRs) in low passage dengue3 viruses (DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution (1977-2000) comparing to the DEN3prototype ( H87). Methods: The 5’UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced. The multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software. Replication of five Thai DEN3 candidates comparing done to human (HepG2) and the mosquito (C6 / 36) cell lines. Results: Among these Thai DEN3, the completely identical sequences of their first 89 nucleotides, their high -order secondary structure of 5’UTRs and three SNPs including the predominant C90 T, and two minor SNPs including A109 G and Al12G were found. C90 T of Thai DEN3.Bangkok isolates was shown predominantly before 1977. Feive Thai DEN3 candidates with the predominant C90 T were shown to replicate in human (HepG2) and the mosquito (C6 / 36) cell lines better than the H87 prototype. Yet, their highly conserved sequences as well as SNPs of the 5 ’UTR did not appear to correlate with their disease severity in human. Conclusions: Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence, the high-order secondary structure and the predominant C90 T of the 5’UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.