Background Pakistan is among three countries endemic for wild poliovirus (WPV1) circulation, still struggling for eradication of poliomyelitis. Active clinical and environmental surveillance systems with meticulous laboratory investigations provide insights into poliovirus transmission patterns and genomic diversity to inform decisions for strategic operations required to achieve eradication. Methods We analyzed epidemiological and virological data at molecular level to comprehend the current epidemiological status of WPV1 in Pakistan during 2015-2017. Stool specimens of AFP patients and sewage samples collected from 60 active environmental sites. Viral culturing, intratypic differentiation by real time-PCR and nucleic acid sequencing of VP1 region of poliovirus genome to determine the genetic relatedness among WPV1 strains were applied. The phylogenetic analysis were done using BEAST v2.3.0 [1] . Results Poliovirus isolates were grouped into eleven distinct clusters which had ≥95% nucleotide homology in VP1 coding region. Most burden of poliovirus was shared by three major reservoirs i.e. Karachi, Peshawar and Quetta block (64.2% in 2015; 75.4% in 2016 and 76.7% in 2017). Conclusions Environmental surveillance reveals importations and pockets of unimmunized children which dictate intensive target mop-up campaigns in such areas to contain poliovirus transmission. Decrease in number of orphan isolates reflects effective combination of AFP and ES surveillance working in Pakistan. The genetic data reflects sustained transmission within reservoir areas, further expanded by periodic importations to areas of high immunity reflected by immediate termination of imported viruses. However, it is suggestive that Pakistan is at-risk unless the entire country including Afghanistan attain a polio-free status. Improved immunization coverage with high quality surveillance is vital for global certification.

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