Background Even though dengue has been recognized as one of the major public health threats in Pakistan the understanding of its molecular epidemiology is still limited. (47%) sera XL147 by a polymerase chain reaction assay. These included 36 (38.3%) DENV-2 57 DENV-3 (60.6%) and 1 DENV-4 (1.1%) cases. Sequences of 13 whole genomes (6 DENV-2 6 DENV-3 and 1 DENV-4) and 49 envelope genes (26 DENV-2 22 DENV-3 and 1 DENV-4) were analysed to determine the origin phylogeny diversity and selection pressure during virus evolution. Results DENV-2 DENV-3 and DENV-4 in Pakistan from 2006 to 2011 shared 98.5-99.6% nucleotide XL147 and 99.3-99.9% amino acid similarity with those circulated in the Indian subcontinent during the last decade. Nevertheless Pakistan DENV-2 and DENV-3 strains formed distinct clades characterized by amino XL147 acid signatures of NS2A-I116T + NS5-K861R and NS3-K590R + NS5-S895L respectively. Each clade consisted of a heterogenous virus population that circulated in Southern (2006-2009) and Northern Pakistan (2011). Conclusions DENV-2 DENV-3 and DENV-4 that circulated during 2006-2011 are likely to have first introduced via the southern route of Pakistan. Both DENV-2 and DENV-3 have undergone in-situ evolution to generate heterogenous populations possibly driven by sustained local DENV transmission during 2006-2011 periods. While both DENV-2 and DENV-3 continued to circulate in Southern Pakistan until 2009 DENV-2 has spread in a Northern direction to establish in Punjab Province which experienced a massive dengue outbreak in 2011. and The transmission of DENV has increased in recent years in urban and semi-urban endemic settings especially in Americas South and South-east Asia and the Western ATV Pacific. The magnitude distribution and clinical severity of dengue outbreaks have been alarmingly high in countries such as India  Sri Lanka  Nepal  Bangladesh  and Pakistan  in the Indian subcontinent during the last decade. Pakistan is usually endemic to all four serotypes of DENV circulating throughout the year with a peak incidence during the post monsoon period between October and December [7 8 Factors such as crowded cities unsafe drinking water inadequate sanitation and large number of refugees facilitate the spread of dengue in different parts of the country resulting in increased morbidity and mortality. It is believed that DENV was first introduced into Pakistan through the importation of tyres made up of eggs of infected mosquitoes at Karachi sea port . Although serological evidence of DENV infections in Pakistan dates back to 1968  the first confirmed outbreak associated with DHF occurred in the southern Pakistan city of Karachi in 1994 . Serological studies confirmed the circulation of both DENV-1 and DENV-2 during the 1994 outbreak . Since then the disease has emerged as a major public health problem in the country . DENV-3 was first reported during the 2005-2006 outbreak in Karachi [14 15 By 2007 dengue started to emerge in the Northern Pakistan. DENV-2 and DENV-3 have been the dominant serotypes in Lahore from 2007 to 2009 . Dengue showed a resurgence in November 2010 especially in Sindh Punjab and Khyber Pakhtunkhwa regions subsequent to massive floods in July same year . The outbreak escalated in Lahore Punjab in 2011 with 22 562 cases and 363 deaths due to severe DHF . So far there have been a few comprehensive molecular epidemiological studies that describe DENV circulating in Pakistan. There is only one genome-wide analysis that describes Pakistani DENV-2 circulated during the 2011 outbreak . The studies of molecular epidemiology and evolutionary genetics are important to predict the origin and spread of viruses to strengthen our understanding around the pathogenesis of disease the cause of epidemics and XL147 the genetic basis of virulence. RNA viruses such as DENV evolve rapidly  and on rare occasions certain mutations lead to phenotypic changes in the viruses that alter their potential to cause outbreaks associated XL147 with severe disease . Genotypic characterization has been a useful tool in determining the evolutionary origin of the DENV identifying the circulating virus strains in an endemic area and detecting the introduction of new genotypes. In recent years envelope (E) gene sequencing has been widely used to assess the phylogenetic relationships among DENV isolates . In Pakistan sequencing of NS3 gene .