Abstract: Background and Objective: Dengue is the most important arthropod-borne viral disease which caused by the four-dengue virus serotypes (1-4) and its incidence has grown dramatically around the world in the recent decades. This study was conducted to determine the molecular characterization of dengue virus genotypes spreading in Jeddah province. Methodology: To distinguish dengue virus genotypes, serum samples from 13 infected patients were subjected to envelop and non-structural 1 (E/NS1) gene amplification and sequence analysis at the nucleotide and amino acid levels. Results: The present partial dengue virus phylogenetic analysis announced the domination of dengue virus 2 genotype among the current dengue virus samples circulating in Jeddah province. Dengue virus 2 current isolates were grouped in one branch and seemed to be more closely related to various strains isolated from Sri-Lanka, Australia and Singapore and confirmed by internucleotide distance average ranged +/-0.01. Interestingly, sequences analysis of amino acids confirmed substitution of 8 amino acid residue (Ser729Gua, Ser729Arg, Val762Gau, Val780phe, Val781Leu, Val781Ala, Glu858Asp and Gln873His) among the present isolates comparing with previous references strains isolated from different countries. Remarkably, one unique amino acid residue Ala741Val was verified in the 10 present isolates compared to the reference sequence previously isolated from Jeddah. Conclusion: Notably, the present study demonstrated the sequencing analysis of the dengue virus 2-E/NS1 on both nucleotide and amino acid levels and confirmed its endogenously prevalence in Jeddah.