The dengue is the more important arboviruses which afect the humans in terms of mortality and morbidity, characterized as febrile Illness acute autolimited, which the etiologic agent related to Flavivirus genus. The Rondonia State is located in Amazon Region, north of Brazil, being a considered State triple favorable the propagation of the dengue virus, therefore is connected by road to others parts of country; for being border with another country and still to exist population migrationsThe dengue virus identification and molecular characterization are important for serotyping determination circulating in the region, the source of virus and the correlation of virulence and epidemiology. This dissertation has as objective of standardize of techniques for identification and molecular characterization of dengue virus in urban and peri-urbans areas from Porto Velho.Collected blood samples of patients with clinical suspects of dengue fever between the months of January/2001 and December/2003. Isolate the virus using the C6/36 cells and confirmed by immufluorescence assay. The RNA viral was extracted for two methods: the TRIzol® and QUIAamp RNA viral mini kit ®. After, submitted the extracted RNA to RT-PCR with two different pairs of primers. The first pairs, AD3 and AD4 identify the dengue virus and the serotyping characterization was made with restriction enzymes Kpn I and Hind III, for dengue virus serotype 1 and 2 respectively. Again, for identification of dengue virus used the primers D1 and D2, following of a Hemi-nested-PCR for serotyping characterization of dengue virus type 1, 2, 3 and 4. One hundred and fifteen blood samples were collected of patients. Seventy were positive in immunofluorescence assay. Forty sample were extracted RNA viral by TRIzol® an 30 samples by QIAamp RNA mini kit ®. Sixty were characterized as dengue virus type 1 for restriction enzymes method. Teen sample were characterized by Hemi-nested-PCR as dengue virus type 3 This study showed which the dengue virus type 1 and 3 circulate in Porto Velho; was possible standardized the technique for RNA viral extraction for RT-PCR directly of serum samples and establishment of techniques for epidemiological surveillance. The sensibility of RNA extraction directly of serum samples was relation with the viremia.