Malaria transmission has been significantly reduced through the past decades via intensive, multifaceted control efforts. However, challenges to elimination remain in the form of residual asymptomatic and imported infections increasing the risk of resurgence. Declining transmission limits genetic recombination in circulating parasites, leading to fragmented parasite population structure. Human populations are then exposed to fewer, possibly only local circulating parasites, which potentially limits naturally acquired immunity to these strains. Consequently, this may increase host susceptibility to clinical symptoms if infected with introduced strains. Additionally, asymptomatic infections appear more prevalent in low-transmission regions and often undetected, serve as reservoirs for ongoing transmission. The interplay between decreasing transmission, parasite diversity, and host immunity may contribute to prevalence of asymptomatic and symptomatic infections. Papua New Guinea (PNG), historically a region of high malaria transmission, recorded substantial transmission decline (2012) followed by resurgence of infection (2016), providing an opportunity to evaluate parasite population structure and sources of resurgence at low-transmission. Utilizing asymptomatic isolates from cross-sectional surveys in East Sepik, PNG in 2012 and 2016, and clinical isolates from a therapeutic efficacy study in 2012, we assessed Plasmodium falciparum strain diversity and investigated the association between parasite genetics and symptomatic malaria in 2012, and resurgent lineages in 2016. P. falciparum infections were compared using SNP barcoding and ‘varcoding’ of the var genes encoding the major surface antigen to evaluate parasite diversity, relatedness, population structure and antigenic diversity. Clustering (PCA), nucleotide diversity (Hierfstat), pairwise relatedness (IsoRelate) and logistic regression were used to identify origins of infections, transmission chains, links between infections and strain diversity, proportion of imported and local strains, and its variation with transmission levels. These results aim to fill a crucial knowledge gap regarding sources of infections in 2012 and resurgent cases in 2016, contributing to improved surveillance and outbreak prediction in malaria-endemic regions.