The prevalence of all four dengue virus (DENV) serotypes has increased dramatically in recent years in many tropical and sub-tropical countries accompanied by an increase in genetic diversity within each serotype. This expansion in genetic diversity is expected to give rise to viruses with altered antigenicity, virulence, and transmissibility. We previously demonstrated the co-circulation of multiple DENV genotypes in Thailand and identified a predominant genotype for each serotype. In this study, we performed a comparative analysis of the complete genomic sequences of 28 DENV-3 predominant genotype II strains previously collected during different DENV-3 epidemics in Thailand from 1973 to 2001 with the goal to define mutations that might correlate with virulence, transmission frequency, and epidemiological impact. The results revealed (1) 37 amino acid and six nucleotide substitutions adopted and fixed in the virus genome after their initial substitutions over nearly 30-year-sampling period, (2) the presence of more amino acid and nucleotide substitutions in recent virus isolates compared with earlier isolates, (3) six amino acid substitutions in capsid (C), pre-membrane (prM), envelope (E), and nonstructural (NS) proteins NS4B and NS5, which appeared to be associated with periods of high DENV-3 epidemic activity, (4) the highest degree of conservation in C, NS2B and the 5'-untranslated region (UTR), and (5) the highest percentage of amino acid substitutions in NS2A protein.