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Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host. Author summary: Dengue virus (DENV) is an RNA virus which causes many infections worldwide. Taiwan experienced the largest DENV-2 outbreak in 2015 which resulted in many fatalities. Specific reasons for the increase in severity during this outbreak remains unclear. The molecular epidemiology of this outbreak showed that clade replacement may have occurred, with the 2015 isolates forming a new clade and the old clade consisted of sequences from 2001–2004 in Taiwan. A dominant C-A314G (K73R) substitution was found in the 2015 DENV-2 outbreak isolates, which may have resulted in a clade replacement in the DENV-2 Cosmopolitan genotype. Further analysis of genetic variation of the virus population of one DENV-2 infected patient showed a decrease of haplotypes during the progression of infection and an NS4A-L115I mutation that did not appear until a later time-point. These show the difference between the variations found in a population wide analysis compared to an infrapopulation analysis which can influence viral infection. [ABSTRACT FROM AUTHOR]