We studied the evolution, genotypes, and the molecular clock of dengue virus serotype 1 (DENV-1), between 2001 and 2014 in Guangzhou, China. The analysis of the envelope (E) gene sequences of 67 DENV-1 strains isolated in Guangzhou, together with 58 representative sequences downloaded from NCBI, have shown shifts in viral genotypes. The genotype changed several times, from genotype I to IV in 2002, from IV to I in 2005, and from I to V in 2014. These genotype shifts may be the cause of DENV outbreaks. The diversity of genotypes and clades demonstrates a high risk of future outbreaks in Guangzhou. The mean rate of virus nucleotide substitution in Guangzhou was determined to be 7·77 × 10−4 per site per year, which represents a medium substitution rate compared to two other countries. Our research can point to different ancestors of the isolated strains, which may further reveal the different origins and transmission of DENV-1 strains in Guangzhou.